Transposition of nucleic acid constructs into eukaryotic genomes with a transposase from amyelois

ABSTRACT

The present invention provides polynucleotide vectors for high expression of heterologous genes. Some vectors further comprise novel transposons and transposases that further improve expression. Further disclosed are vectors that can be used in a gene transfer system for stably introducing nucleic acids into the DNA of a cell. The gene transfer systems can be used in methods, for example, gene expression, bioprocessing, gene therapy, insertional mutagenesis, or gene discovery.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 62/831,097 filed Apr. 8, 2019; U.S. Provisional Application No.62/873,342 filed Jul. 12, 2019; and U.S. Provisional Application No.62/990,568 filed Mar. 17, 2020, each incorporated by reference in itsentirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

The application refers to sequences disclosed in a txt file named546905SEQLST.txt, of 2,392,166 bytes, created Apr. 6, 2020, incorporatedby reference.

1. FIELD OF THE INVENTION

The field of the present invention relates to configurations of DNAvectors for making stable modifications of the genomes of target cells,and the use of non-natural transposons and transposases.

2. BACKGROUND OF THE INVENTION

The expression levels of genes encoded on a polynucleotide integratedinto the genome of a cell depend on the configuration of sequenceelements within the polynucleotide. The efficiency of integration andthus the number of copies of the polynucleotide that are integrated intoeach genome, and the genomic loci where integration occurs alsoinfluence the expression levels of genes encoded on the polynucleotide.The efficiency with which a polynucleotide may be integrated into thegenome of a target cell can often be increased by placing thepolynucleotide into a transposon.

Transposons comprise two ends that are recognized by a transposase. Thetransposase acts on the transposon to remove it from one DNA moleculeand integrate it into another. The DNA between the two transposon endsis transposed by the transposase along with the transposon ends.Heterologous DNA flanked by a pair of transposon ends, such that it isrecognized and transposed by a transposase is referred to herein as asynthetic transposon. Introduction of a synthetic transposon and acorresponding transposase into the nucleus of a eukaryotic cell mayresult in transposition of the transposon into the genome of the cell.These outcomes are useful because they increase transformationefficiencies and because they can increase expression levels fromintegrated heterologous DNA. There is thus a need in the art forhyperactive transposases and transposons.

Transposition by a piggyBac-like transposase is perfectly reversible.The transposon is initially integrated at an integration target sequencein a recipient DNA molecule, during which the target sequence becomesduplicated at each end of the transposon inverted terminal repeats(ITRs). Subsequent transposition removes the transposon and restores therecipient DNA to its former sequence, with the target sequenceduplication and the transposon removed. However, this is not sufficientto remove a transposon from a genome into which it has been integrated,as it is highly likely that the transposon will be excised from thefirst integration target sequence but transposed into a secondintegration target sequence in the genome. Transposases that aredeficient for the integration (or transposition) function, on the otherhand, can excise the transposon from the first target sequence, but willbe unable to integrate into a second target sequence.Integration-deficient transposases are thus useful for reversing thegenomic integration of a transposon.

One application for transposases is for the engineering of eukaryoticgenomes. Such engineering may require the integration of more than onedifferent polynucleotide into the genome. These integrations may besimultaneous or sequential. When transposition into a genome of a firsttransposon comprising a first heterologous polynucleotide by a firsttransposase is followed by transposition into the same genome of asecond transposon comprising a second heterologous polynucleotide by asecond transposase, it is advantageous that the second transposase notrecognize and transpose the first transposon. This is because thelocation of a polynucleotide sequence within the genome influences theexpressibility of genes encoded on said polynucleotide, so transpositionof the first transposon to a different chromosomal location by thesecond transposase could change the expression properties of any genesencoded on the first heterologous polynucleotide. There is therefore aneed for a set of transposons and their corresponding transposases inwhich the transposases within the set recognize and transpose only theircorresponding transposons, but not any other transposons in the set.

Since its discovery in 1983, the piggyBac transposon and transposasefrom the looper moth Trichoplusia ni has been widely used for insertingheterologous DNA into the genomes of target cells from many differentorganisms. The piggyBac system is a particularly valuable transposasesystem because of: “its activity in a wide range of organisms, itsability to integrate multiple large transgenes with high efficiency, theability to add domains to the transposase without loss of activity, andexcision from the genome without leaving a footprint mutation” (Dohertyet al., Hum. Gene Ther. 23, 311-320 (2012), at p. 312, LHC, ¶2).

The value and versatility of the piggyBac system has inspiredsignificant efforts to identify other active piggyBac-like transposons(commonly referred to as piggyBac-like elements, or PLEs) but these havebeen largely unsuccessful. “Since piggyBac is one of the most populartransposons used for transgenesis, searching for new active PLEs hasattracted lots of attention. However, only a few active PLEs have beenreported to date.” (Luo et al., BMC Molecular Biology 15, 28 (2014)worldwide web biomedcentral.com/1471-2199/15/28. p.4 of 12, RHC, ¶1“Discussion”).

Although there are large numbers of homologs of piggyBac transposons andtransposases in sequence databases, few active ones have been identifiedbecause the vast majority are inactivated by their hosts to avoidactivity deleterious to the hosts as illustrated by the followingexcerpts: “Related piggyBac transposable elements have been found inplants, fungi and animals, including humans [125], although they areprobably inactive due to mutation.” (Munoz-Lopez & Garcia-Perez, CurrentGenomics 11, 115-128 (2010) at p. 120, RHC, ¶1). “It is believed thattransposons invade a genome and subsequently spread throughout it duringevolution. The “selfish” mobility of transposons is harmful to the host;hence, they are eliminated or inactivated by the host through naturalselection. Even harmless transposons lose the activity eventuallybecause of the absence of conservative selection for them. Thus, ingeneral, transposons have a short life span in a host and theysubsequently become fossils in the genome.” (Hikosaka et al., Mol. Biol.Evol, 24, 2648-3656 (2007) at p. 2648, LHC, ¶1 “Introduction”).“Frequent movement of transposable elements in a genome is harmful(Belancio et al., 2008; Deininger & Batzer, 1999; Le Rouzic & Capy,2006; Oliver & Greene, 2009). As a result, most transposable elementsare inactivated shortly after they invade a new host.” (Luo et al.,Insect Science 18, 652-662 (2011) at p. 660, LHC, ¶1).

Three classes of piggyBac-like elements have been found: (1) those thatare very similar to the original piggyBac from the looper moth(typically >95% identical at the nucleotide level), (2) those that aremoderately related (typically 30-50% identical at the amino acid level),and (3) those that are very distantly related (Wu et al., Insect Science15, 521-528 (2008) at p. 521, RHC. ¶2).

PiggyBac-like transposases highly related to the looper moth transposasehave been described by several groups. They are extremely highlyconserved. Very similar transposase sequences to the original piggyBac(95-98% nucleotide identity) have been reported in three differentstrains of the fruit fly Bactrocera dorsalis (Handler & McCombs, InsectMolecular Biology 9, 605-612, (2000)). Comparably conserved piggyBacsequences have been found in other Bactrocera species (Bonizzoni et al.,Insect Molecular Biology 16, 645-650 (2007)). Two species of noctuidmoth (Helicoverpa zea and Helicoverpa armigera) and other strains of thelooper moth Trichoplusia ni had genomic copies of the piggyBactransposase with 93-100% nucleotide identity to the original piggyBacsequence (Zimowska & Handler, Insect Biochemistry and Molecular Biology,36, 421-428 (2006)). Zimowska & Handler also found multiple copies ofmuch more significantly mutated (and truncated) versions of the piggyBactransposase in both Helicoverpa species, as well as a homolog in thearmyworm Spodptera frugiperda. None of these groups attempted to measureany activity for these transposases. Wu et. al (2008), supra, reportedisolating a transposase from Macdunnoughia crassisigna with 99.5%sequence identity with the looper moth piggyBac. They also demonstratedthat this transposon and transposase are active, by showing that theycould measure both excision and transposition. Their Discussionsummarized previous results as follows: “Other reportedly closelyrelated IFP2 class sequences were in various Bactrocera species, T. nigenome, Heliocoverpa armigera, and H. zea (Handler & McCombs, 2000;Zimowska & Handler, 2006; Bonizzoni et al., 2007). These sequences werepartial fragments of piggyBac-like elements, and most of them weretruncated or inactivated by accumulating random mutations.” (Wu et. al.,Insect Science 15, 521-528 (2008) at p. 526, LHC, ¶3.)

It has proved very difficult to identify active piggyBac-liketransposases that are moderately related to the looper moth enzymesimply by looking at sequence. The presence of features that are knownto be necessary: a full-length open reading frame, catalytic aspartateresidues and intact ITRs, has not proven to be predictive of activity.“A large diversity of PLEs in eukaryotes has been documented in acomputational analysis of genomic sequence data [citations omitted].However, few elements were isolated with an intact structure consistentwith function, and only the original IFP2 piggyBac has been developedinto a vector for routine transgenesis.” (Wu et al., Genetica 139,149-154 (2011), at p. 152, RHC, ¶2.). Wu et al.'s group from NanjingUniversity (the “Nanjing group”) published several papers over a 6-yearperiod, each identifying moderately related piggyBac homologs. Althoughthe Nanjing group showed in 2008 that they could measure both excisionand transposition of the Macdunnoughia crassisigna transposon by itscorresponding transposase, and in each subsequent paper they express thedesire to identify novel active piggyBac-like transposases, they onlyshow excision activity and that only for one transposase from Aphisgossypii. They conclude that the usefulness of this transposase “remainsto be explored with further experiments” (Luo et. al. 2011, p. 660, LHC¶2 “Discussion”). However, none of the other papers published by theNanjing group in which piggyBac-like sequences were identified from avariety of other insects, show that any activity was found. Three papersidentifying other putative active piggyBac-like transposases werepublished by a group at Kansas State University. None of these papersreports any activity data. Wang et al., Insect Molecular Biology 15,435-443 (2006) found multiple copies of piggyBac-like sequences in thegenome of the tobacco budworm Heliothis virescens. Many of these hadobvious mutations or deletions that led the authors not to consider themto be candidate active transposases. Wang et. al., Insect Biochemistryand Molecular Biology 38, 490-498 (2008) reported more than 30piggyBac-like sequences in the genome of the red flour beetle Triboliumcastaneum. They concluded “All the TcPLEs identified here, exceptTcPLE1, were apparently defective due to the presence of multiple stopcodons and/or indels in the putative transposase encoding regions.” Evenfor TcPLE1 there was “no evidence supporting recent or currentmobilization events” (p. 492, section 3.1, IT 2&3). Wang et al. (2010)used PCR to identify piggyBac-like sequences from the pink bollwormPectinophora gossypiella. Again, they found many obviously defectivecopies, as well as one transposase with characteristics the authorsbelieve to be consistent with activity (page 179, RHC, ¶2). But nofollow up report indicating transposase activity can be found. Othergroups have also attempted to identify active piggyBac-liketransposases. These reports conclude with statements that thepiggyBac-like elements identified are undergoing testing for activity,but there are no subsequent reports of success. For example, Sarkar et.al. (2003) conclude their Discussion by re-stating the value of novelactive piggyBac-like transposons, and describing their ongoing effortsto identify one: “The mobility of the original T. ni piggyBac element invarious insects suggests that piggyBac family transposons might prove tobe useful genetic tools in organisms other than insects. We arecurrently isolating an intact piggyBac element from An. gambiae (AgaPB1)to test its mobility in various organisms.” (Mol. Gen. Genomics 270,173-180 at p. 179, LHC, ¶1). There appear to be no further publishedreports of this putative active transposase. Xu et al. analyzed thesilkworm genome looking for piggyBac-like sequences (Xu et al., Mol GenGenomics 276, 31-40 (2006)). They found 98 piggyBac-like sequences andperformed various computational analyses of putative transposasesequence and ITR sequences. They conclude: “We have isolated severalintact piggyBac-like elements from B. mori and are currently testingtheir activity and the feasibility of using them as transformationvectors.” (p 38, RHC, ¶3). There appear to be no further publishedreports of these putative active transposases.

Four published papers discussing the third class of distantly relatedpiggyBac-like transposases. The first three of these demonstrate onlythe excision part of the reaction and acknowledge that this is differentfrom full transposition. Hikosaka et. al., Mol Biol Evol 24, 2648-2656(2007) reported that “In the present study, we demonstrated that theXtr-Uribo2 Tpase has excision activity toward the target transposon,although there is no evidence for the integration of the excised targetinto the genome thus far.” (page 2654, RHC, ¶2). Luo et. al., InsectScience 18, 652-662 (2011) reported “These results demonstrated theactivity of the Ago-PLE1.1 transposase in mediating the first step ofthe cut and -paste movement of the element” (page 658, LHC, ¶1). Daimonet. al., Genome 53, 585-593 (2010) discussed the transposase systemsyabusabe-1 and yabusabe-W. Although Daimon et al. reported detecting anexcision event by PCR, they also report screening approximately 100,000recovered plasmids for the excision of yabusame-1 and yabusame-W withoutidentifying a single recovered plasmid from which the elements hadexcised. By contrast Daimon reports the transposition frequency ofwildtype piggyBac enzyme as around 0.3-1.4. Thus, it appears from Daimonet al. that the excision frequency of yabusabe-1 or -W is less than0.001% (1:100,000). This is at least 2-3 orders of magnitude less thancan be achieved with a wildtype piggyBac enzyme and even less thanavailable genetically engineered variants of piggyBac transposase, whichachieve ten-fold higher transposition than wildtype. The impliedtransposition frequency for yabasume-1 from Daimon et al. is also twoorders of magnitude lower than random integration frequency in mammaliancells (which is of the order of 0.1%). Thus, Daimon et al. show thatyabusame-1 was essentially inactive and would not be useful as a geneticengineering tool. Such a view likely underlies Daimon et al.'s ownconclusion: “Although we could detect the excision event in the highlysensitive PCR-based assay, our data indicate that both elements havelost their excision activity almost entirely.” This also suggests thatthe PCR-based excision assay used to show activity of Uribo2 andAgo-PLE1.1 is not predictive of transposition activity that will beuseful for inserting heterologous DNA into the genome of a target cell.The only report of a fully active piggyBac-like transposase (competentfor both excision and integration) of the third category of distantlyrelated transposases to the original piggyBac transposase fromTrichoplusia Ni is one from the bat Myotis lucifugus (Mitra et. al.,Proc. Natl. Acad. Sci. 110, 234-239 (2013)). These authors used a yeastsystem to demonstrate both excision and transposition activities for thebat transposase. All of the work described here shows that it has beenextremely difficult to identify fully active piggyBac-like transposases,even though there are a large number of candidate sequences. There istherefore a need for new piggyBac-like transposons and theircorresponding transposases.

3. SUMMARY OF THE INVENTION

Heterologous gene expression from polynucleotide constructs that stablyintegrate into a target cell genome can be improved by placing theexpression polynucleotide between a pair of transposon ends: sequenceelements that are recognized and transposed by transposases. DNAsequences inserted between a pair of transposon ends can be excised by atransposase from one DNA molecule and inserted into a second DNAmolecule. A novel piggyBac-like transposon-transposase system isdisclosed that is not derived from the looper moth Trichoplusia ni. Itis derived from the naval orangeworm moth Amyelois transitella (theAmyelois transposase). The Amyelois transposon comprises sequences thatfunction as transposon ends and that can be used in conjunction with acorresponding Amyelois transposase that recognizes and acts on thosetransposon ends, as a gene transfer system for stably introducingnucleic acids into the DNA of a cell. The gene transfer systems of theinvention can be used in methods including but not limited to genomicengineering of eukaryotic cells, heterologous gene expression, genetherapy, cell therapy, insertional mutagenesis, or gene discovery.

Transposition may be effected using a polynucleotide comprising an openreading frame encoding an Amyelois transposase, the amino acid sequenceof which is at least 90% identical to SEQ ID NO: 18, operably linked toa heterologous promoter. The heterologous promoter may be active in aeukaryotic cell. The heterologous promoter may be active in a mammaliancell. mRNA may be prepared from a polynucleotide comprising an openreading frame encoding an Amyelois transposase, the amino acid sequenceof which is at least 90% identical to SEQ ID NO: 18, operably linked toa heterologous promoter that is active in an in vitro transcriptionreaction. The transposase may comprises a mutation as shown in columns Cand D in Table 1, relative to the sequence of SEQ ID NO: 18. Thetransposase may comprise a mutation at an amino acid position selectedfrom 79, 95, 115, 116, 121, 139, 166, 179, 187, 198, 203, 211, 238, 273,304, 329, 345, 362, 366, 408, 416, 435, 458, 475, 483, 491, 529, 540,560 and 563, relative to the sequence of SEQ ID NO: 18. The transposasemay comprise a mutation selected from D79N, R95S, L115D, E116P, H121Q,K139E, V166F, G179N, W187F, P198R, L203R, N211R, E238D, L273M, L273I,D304R, D304K, Q329G, T345L, K362R, T366R, L408M, S416E, S435G, L458M,V475I, N483K, I491M, A529P, K540R, S560K and S563K, relative to thesequence of SEQ ID NO: 18, the transposase optionally including at least2, 3, 4, or 5 selected from the group. The amino acid sequence of thetransposase may be selected from SEQ ID NO: 96-170. The transposase canexcise or transpose a transposon from SEQ ID NO: 192. The excisionactivity or transposition activity of the transposase is at least 2-foldhigher than the activity of SEQ ID NO: 18, optionally 2 to 10 foldhigher. Codons of the open reading frame of the transposase may beselected for mammalian cell expression. An isolated mRNA may encode apolypeptide, the amino acid sequence of which is at least 90% identicalwith SEQ ID NO: 18, and wherein the mRNA sequence comprises at least 10synonymous codon differences relative to SEQ ID NO: 929, and wherein themRNA sequence comprises at least 10 synonymous codon differencesrelative to SEQ ID NO: 929 at corresponding positions between the mRNAand SEQ ID NO:929, optionally wherein codons in the mRNA at thecorresponding positions are selected for mammalian cell expression. Theopen reading frame encoding the transposase may further encode aheterologous nuclear localization sequence fused to the transposase. Theopen reading frame encoding the transposase may further encode aheterologous DNA binding domain (for example derived from a Crispr Cassystem, or a zinc finger protein, or a TALE protein) fused to thetransposase. A non-naturally occurring polynucleotide may encode apolypeptide, the sequence of which is at least 90% identical to SEQ IDNO: 18.

An Amyelois transposon comprises SEQ ID NO: 9 and SEQ ID NO: 10 flankinga heterologous polynucleotide. The transposon may further comprise asequence at least 90% identical to SEQ ID NO: 13 on one side of theheterologous polynucleotide and a sequence at least 90% identical to SEQID NO: 16 on the other. The heterologous polynucleotide may comprise aheterologous promoter that is active in eukaryotic cells. The promotermay be operably linked to at least one or more of: i) an open readingframe; ii) a nucleic acid encoding a selectable marker; iii) a nucleicacid encoding a counter-selectable marker; iii) a nucleic acid encodinga regulatory protein; iv) a nucleic acid encoding an inhibitory RNA. Theheterologous promoter may comprise a sequence selected from SEQ ID NOs:474-558. The heterologous polynucleotide may comprise a heterologousenhancer that is active in eukaryotic cells. The heterologous enhancermay be selected from SEQ ID NOs: 453-473. The heterologouspolynucleotide may comprise a heterologous intron that is spliceable ineukaryotic cells. The nucleotide sequence of the heterologous intron maybe selected from SEQ ID NO: 561-621. The heterologous polynucleotide maycomprise an insulator sequence. The nucleic acid sequence of theinsulator may be selected from SEQ ID NO: 435-441. The heterologouspolynucleotide may comprise two open reading frames, each operablylinked to a separate promoter. The heterologous polynucleotide maycomprise a sequence selected from SEQ ID NOs: 745-928. The heterologouspolynucleotide may comprise or encode a selectable marker. Theselectable marker may be selected from a glutamine synthetase enzyme, adihydrofolate reductase enzyme, a puromycin acetyltransferase enzyme, ablasticidin acetyltransferase enzyme, a hygromycin B phosphotransferaseenzyme, an aminoglycoside 3′-phosphotransferase enzyme and a fluorescentprotein. A eukaryotic cell whose genome comprises SEQ ID NO: 9 and SEQID NO: 10 flanking a heterologous polynucleotide is an embodiment of theinvention. The cell may be an animal cell, a mammalian cell, a rodentcell or a human cell.

A transposon may be integrated into the genome of a eukaryotic cell by(a) introducing into the cell a transposon comprising SEQ ID NO: 9 andSEQ ID NO: 10 flanking a heterologous polynucleotide, (b) introducinginto the cell a transposase, the sequence of which is at least 90%identical with SEQ ID NO: 18 wherein the transposase transposes thetransposon to produce a genome comprising SEQ ID NO: 9 and SEQ ID NO: 10flanking the heterologous polynucleotide. The transposase may beintroduced as a polynucleotide encoding the transposase, thepolynucleotide may be an mRNA molecule or a DNA molecule. Thetransposase may be introduced as a protein. The heterologouspolynucleotide may also encode a selectable marker, and the method mayfurther comprise selecting a cell comprising the selectable marker. Thecell may be an animal cell, a mammalian cell, a rodent cell or a humancell. The human cell may be a human immune cell, for example a B-cell ora T-cell. The heterologous polynucleotide may encode a chimeric antigenreceptor. A polypeptide may be expressed from the transposon integratedinto the genome of the eukaryotic cell. The polypeptide may be purified.The purified polypeptide may be incorporated into a pharmaceuticalcomposition.

4. BRIEF DESCRIPTION OF FIGURES

FIG. 1. Structure of an Amyelois transposon.

An Amyelois transposon comprises a left transposon end and a righttransposon end flanking a heterologous polynucleotide. The lefttransposon end comprises (i) a left target sequence, which is often5′-TTAA-3′, although a number of other target sequences are used atlower frequency (Li et al., 2013. Proc. Natl. Acad. Sci vol. 110, no. 6,E478-487); (ii) a left ITR (e.g. SEQ ID NO: 9) and (iii) (optionally)additional left transposon end sequences (e.g. SEQ ID NO: 13). The righttransposon end comprises (i) (optionally) additional right transposonend sequences (e.g. SEQ ID NO: 16); (ii) a right ITR (e.g. SEQ ID NO:10) which is a perfect or imperfect repeat of the left ITR, but ininverted orientation relative to the left ITR and (iii) a right targetsequence which is typically the same as the left target sequence.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 Definitions

Use of the singular forms “a,” “an,” and “the” include plural referencesunless the context clearly dictates otherwise. Thus, for example,reference to “a polynucleotide” includes a plurality of polynucleotides,reference to “a substrate” includes a plurality of such substrates,reference to “a variant” includes a plurality of variants, and the like.

Terms such as “connected,” “attached,” “linked,” and “conjugated” areused interchangeably herein and encompass direct as well as indirectconnection, attachment, linkage or conjugation unless the contextclearly dictates otherwise. Where a range of values is recited, it is tobe understood that each intervening integer value, and each fractionthereof, between the recited upper and lower limits of that range isalso specifically disclosed, along with each subrange between suchvalues. The upper and lower limits of any range can independently beincluded in or excluded from the range, and each range where either,neither or both limits are included is also encompassed within theinvention. Where a value being discussed has inherent limits, forexample where a component can be present at a concentration of from 0 to100%, or where the pH of an aqueous solution can range from 1 to 14,those inherent limits are specifically disclosed. Where a value isexplicitly recited, it is to be understood that values which are aboutthe same quantity or amount as the recited value are also within thescope of the invention. Where a combination is disclosed, each subcombination of the elements of that combination is also specificallydisclosed and is within the scope of the invention. Conversely, wheredifferent elements or groups of elements are individually disclosed,combinations thereof are also disclosed. Where any element of aninvention is disclosed as having a plurality of alternatives, examplesof that invention in which each alternative is excluded singly or in anycombination with the other alternatives are also hereby disclosed; morethan one element of an invention can have such exclusions, and allcombinations of elements having such exclusions are hereby disclosed.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,Dictionary of Microbiology and Molecular Biology, 2nd Ed., John Wileyand Sons, New York (1994), and Hale & Marham, The Harper CollinsDictionary of Biology, Harper Perennial, N Y, 1991, provide one of skillwith a general dictionary of many of the terms used in this invention.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are described. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively. The terms defined immediately beloware more fully defined by reference to the specification as a whole.

The “configuration” of a polynucleotide means the functional sequenceelements within the polynucleotide, and the order and direction of thoseelements.

The terms “corresponding transposon” and “corresponding transposase” areused to indicate an activity relationship between a transposase and atransposon. A transposase transposes its corresponding transposon. Manytransposases may correspond with a single transposon, and manytransposons may correspond with a single transposase.

The term “counter-selectable marker” means a polynucleotide sequencethat confers a selective disadvantage on a host cell. Examples ofcounter-selectable markers include sacB, rpsL, tetAR, pheS, thyA,gata-1, ccdB, kid and barnase (Bernard, 1995, Journal/Gene, 162:159-160; Bernard et al., 1994. Journal/Gene, 148: 71-74; Gabant et al.,1997, Journal/Biotechniques, 23: 938-941; Gababt et al., 1998,Journal/Gene, 207: 87-92; Gababt et al., 2000, Journal/Biotechniques,28: 784-788; Galvao and de Lorenzo, 2005, Journal/Appl EnvironMicrobiol, 71: 883-892; Hartzog et al., 2005, Journal/Yeat, 22:789-798;Knipfer et al., 1997, Journal/Plasmid, 37: 129-140; Reyrat et al., 1998,Journal/Infect Immun, 66: 4011-4017; Soderholm et al., 2001,Journal/Biotechniques, 31: 306-310, 312; Tamura et al., 2005,Journal/Appl Environ Microbiol, 71: 587-590; Yazynin et al., 1999,Journal/FEBS Lett, 452: 351-354). Counter-selectable markers oftenconfer their selective disadvantage in specific contexts. For example,they may confer sensitivity to compounds that can be added to theenvironment of the host cell, or they may kill a host with one genotypebut not kill a host with a different genotype. Conditions which do notconfer a selective disadvantage on a cell carrying a counter-selectablemarker are described as “permissive”. Conditions which do confer aselective disadvantage on a cell carrying a counter-selectable markerare described as “restrictive”.

The term “coupling element” or “translational coupling element” means aDNA sequence that allows the expression of a first polypeptide to belinked to the expression of a second polypeptide. Internal ribosomeentry site elements (IRES elements) and cis-acting hydrolase elements(CHYSEL elements) are examples of coupling elements.

The terms “DNA sequence”, “RNA sequence” or “polynucleotide sequence”mean a contiguous nucleic acid sequence. The sequence can be anoligonucleotide of 2 to 20 nucleotides in length to a full lengthgenomic sequence of thousands or hundreds of thousands of base pairs.

The term “expression construct” means any polynucleotide designed totranscribe an RNA. For example, a construct that contains at least onepromoter which is or may be operably linked to a downstream gene, codingregion, or polynucleotide sequence (for example, a cDNA or genomic DNAfragment that encodes a polypeptide or protein, or an RNA effectormolecule, for example, an antisense RNA, triplex-forming RNA, ribozyme,an artificially selected high affinity RNA ligand (aptamer), adouble-stranded RNA, for example, an RNA molecule comprising a stem-loopor hairpin dsRNA, or a bi-finger or multi-finger dsRNA or a microRNA, orany RNA). An “expression vector” is a polynucleotide comprising apromoter which can be operably linked to a second polynucleotide.Transfection or transformation of the expression construct into arecipient cell allows the cell to express an RNA effector molecule,polypeptide, or protein encoded by the expression construct. Anexpression construct may be a genetically engineered plasmid, virus,recombinant virus, or an artificial chromosome derived from, forexample, a bacteriophage, adenovirus, adeno-associated virus,retrovirus, lentivirus, poxvirus, or herpesvirus. Such expressionvectors can include sequences from bacteria, viruses or phages. Suchvectors include chromosomal, episomal and virus-derived vectors, forexample, vectors derived from bacterial plasmids, bacteriophages, yeastepisomes, yeast chromosomal elements, and viruses, vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, cosmids and phagemids. An expressionconstruct can be replicated in a living cell, or it can be madesynthetically. For purposes of this application, the terms “expressionconstruct”, “expression vector”, “vector”, and “plasmid” are usedinterchangeably to demonstrate the application of the invention in ageneral, illustrative sense, and are not intended to limit the inventionto a particular type of expression construct.

The term “expression polypeptide” means a polypeptide encoded by a geneon an expression construct.

The term “expression system” means any in vivo or in vitro biologicalsystem that is used to produce one or more gene product encoded by apolynucleotide.

A “gene” refers to a transcriptional unit including a promoter andsequence to be expressed from it as an RNA or protein. The sequence tobe expressed can be genomic or cDNA among other possibilities. Otherelements, such as introns, and other regulatory sequences may or may notbe present.

A “gene transfer system” comprises a vector or gene transfer vector, ora polynucleotide comprising the gene to be transferred which is clonedinto a vector (a “gene transfer polynucleotide” or “gene transferconstruct”). A gene transfer system may also comprise other features tofacilitate the process of gene transfer. For example, a gene transfersystem may comprise a vector and a lipid or viral packaging mix forenabling a first polynucleotide to enter a cell, or it may comprise apolynucleotide that includes a transposon and a second polynucleotidesequence encoding a corresponding transposase to enhance productivegenomic integration of the transposon. The transposases and transposonsof a gene transfer system may be on the same nucleic acid molecule or ondifferent nucleic acid molecules. The transposase of a gene transfersystem may be provided as a polynucleotide or as a polypeptide.

Two elements are “heterologous” to one another if not naturallyassociated. For example, a nucleic acid sequence encoding a proteinlinked to a heterologous promoter means a promoter other than that whichnaturally drives expression of the protein. A heterologous nucleic acidflanked by transposon ends or ITRs means a heterologous nucleic acid notnaturally flanked by those transposon ends or ITRs, such as a nucleicacid encoding a polypeptide other than a transposase, including anantibody heavy or light chain. A nucleic acid is heterologous to a cellif not naturally found in the cell or if naturally found in the cell butin a different location (e.g., episomal or different genomic location)than the location described.

The term “host” means any prokaryotic or eukaryotic organism that can bea recipient of a nucleic acid. A “host,” as the term is used herein,includes prokaryotic or eukaryotic organisms that can be geneticallyengineered. For examples of such hosts, see Maniatis et al., MolecularCloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1982). As used herein, the terms “host,” “host cell,”“host system” and “expression host” can be used interchangeably.

A “hyperactive” transposase is a transposase that is more active thanthe naturally occurring transposase from which it is derived.“Hyperactive” transposases are thus not naturally occurring sequences.

‘Integration defective’ or “transposition defective” means a transposasethat can excise its corresponding transposon, but that integrates theexcised transposon at a lower frequency into the host genome than acorresponding naturally occurring transposase.

An “IRES” or “internal ribosome entry site” means a specialized sequencethat directly promotes ribosome binding, independent of a cap structure.

An ‘isolated’ polypeptide or polynucleotide means a polypeptide orpolynucleotide that has been either removed from its naturalenvironment, produced using recombinant techniques, or chemically orenzymatically synthesized. Polypeptides or polynucleotides of thisinvention may be purified, that is, essentially free from any otherpolypeptide or polynucleotide and associated cellular products or otherimpurities.

The terms “nucleoside” and “nucleotide” include those moieties whichcontain not only the known purine and pyrimidine bases, but also otherheterocyclic bases which have been modified. Such modifications includemethylated purines or pyrimidines, acylated purines or pyrimidines, orother heterocycles. Modified nucleosides or nucleotides can also includemodifications on the sugar moiety, for example, where one or more of thehydroxyl groups are replaced with halogen, aliphatic groups, or isfunctionalized as ethers, amines, or the like. The term “nucleotidicunit” is intended to encompass nucleosides and nucleotides.

An “Open Reading Frame” or “ORF” means a portion of a polynucleotidethat, when translated into amino acids, contains no stop codons. Thegenetic code reads DNA sequences in groups of three base pairs, whichmeans that a double-stranded DNA molecule can read in any of sixpossible reading frames-three in the forward direction and three in thereverse. An ORF typically also includes an initiation codon at whichtranslation may start.

The term “operably linked” refers to functional linkage between twosequences such that one sequence modifies the behavior of the other. Forexample, a first polynucleotide comprising a nucleic acid expressioncontrol sequence (such as a promoter, IRES sequence, enhancer or arrayof transcription factor binding sites) and a second polynucleotide areoperably linked if the first polynucleotide affects transcription and/ortranslation of the second polynucleotide. Similarly, a first amino acidsequence comprising a secretion signal or a subcellular localizationsignal and a second amino acid sequence are operably linked if the firstamino acid sequence causes the second amino acid sequence to be secretedor localized to a subcellular location.

The term “orthogonal” refers to a lack of interaction between twosystems. A first transposon and its corresponding first transposase anda second transposon and its corresponding second transposase areorthogonal if the first transposase does not excise or transpose thesecond transposon and the second transposase does not excise ortranspose the first transposon.

The term “overhang” or “DNA overhang” means the single-stranded portionat the end of a double-stranded DNA molecule. Complementary overhangsare those which will base-pair with each other.

A “piggyBac-like transposase” means a transposase with at least 20%sequence identity as identified using the TBLASTN algorithm to thepiggyBac transposase from Trichoplusia ni (SEQ ID NO: 17), and as morefully described in Sakar, A. et. al., (2003). Mol. Gen. Genomics 270:173-180. “Molecular evolutionary analysis of the widespread piggyBactransposon family and related ‘domesticated’ species”, and furthercharacterized by a DDE-like DDD motif, with aspartate residues atpositions corresponding to D268, D346, and D447 of Trichoplusia nipiggyBac transposase on maximal alignment. PiggyBac-like transposasesare also characterized by their ability to excise their transposonsprecisely with a high frequency. A “piggyBac-like transposon” means atransposon having transposon ends which are the same or at least 80% andpreferably at least 90, 95, 96, 97, 98 or 99% or 100% identical to thetransposon ends of a naturally occurring transposon that encodes apiggyBac-like transposase. A piggyBac-like transposon includes aninverted terminal repeat (ITR) sequence of approximately 12-16 bases ateach end, and is flanked on each side by a 4 base sequence correspondingto the integration target sequence which is duplicated on transposonintegration (the Target Site Duplication or Target Sequence Duplicationor TSD). PiggyBac-like transposons and transposases occur naturally in awide range of organisms including Argyrogramma agnate (GU477713),Anopheles gambiae (XP_312615; XP_320414; XP_310729), Aphis gossypii(GU329918), Acyrthosiphon pisum (XP_001948139), Agrotis ypsilon(GU477714), Bombyx mori (BAD11135), Ciona intestinalis (XP_002123602),Chilo suppressalis (JX294476), Drosophila melanogaster (AAL39784),Daphnia pulicaria (AAM76342), Helicoverpa armigera (ABS18391), Homosapiens (NP 689808), Heliothis virescens (ABD76335), Macdunnoughiacrassisigna (EU287451), Macaca fascicularis (AB179012), Mus musculus(NP_741958), Pectinophora gossypiella (GU270322), Rattus norvegicus(XP_220453), Tribolium castaneum (XP_001814566) and Trichoplusia ni(AAA87375) and Xenopus tropicalis (BAF82026), although transpositionactivity has been described for almost none of these.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used interchangeably to refer to a polymericform of nucleotides of any length, and may comprise ribonucleotides,deoxyribonucleotides, analogs thereof, or mixtures thereof. This termrefers only to the primary structure of the molecule. Thus, the termincludes triple-, double- and single-stranded deoxyribonucleic acid(“DNA”), as well as triple-, double- and single-stranded ribonucleicacid (“RNA”). It also includes modified, for example by alkylation,and/or by capping, and unmodified forms of the polynucleotide. Moreparticularly, the terms “polynucleotide,” “oligonucleotide,” “nucleicacid” and “nucleic acid molecule” include polydeoxyribonucleotides(containing 2-deoxy-D-ribose), polyribonucleotides (containingD-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether splicedor unspliced, any other type of polynucleotide which is an N- orC-glycoside of a purine or pyrimidine base, and other polymerscontaining non-nucleotidic backbones, for example, polyamide (forexample, peptide nucleic acids (“PNAs”)) and polymorpholino(commercially available from the Anti-Virals, Inc., Corvallis, Oreg., asNeugene) polymers, and other synthetic sequence-specific nucleic acidpolymers providing that the polymers contain nucleobases in aconfiguration which allows for base pairing and base stacking, such asis found in DNA and RNA. There is no intended distinction in lengthbetween the terms “polynucleotide,” “oligonucleotide,” “nucleic acid”and “nucleic acid molecule,” and these terms are used interchangeablyherein. These terms refer only to the primary structure of the molecule.Thus, these terms include, for example, 3′-deoxy-2′, 5′-DNA,oligodeoxyribonucleotide N3′ P5′ phosphoramidates,2′-O-alkyl-substituted RNA, double- and single-stranded DNA, as well asdouble- and single-stranded RNA, and hybrids thereof including forexample hybrids between DNA and RNA or between PNAs and DNA or RNA, andalso include known types of modifications, for example, labels,alkylation, “caps,” substitution of one or more of the nucleotides withan analog, internucleotide modifications such as, for example, thosewith uncharged linkages (for example, methyl phosphonates,phosphotriesters, phosphoramidates, carbamates, or the like) withnegatively charged linkages (for example, phosphorothioates,phosphorodithioates, or the like), and with positively charged linkages(for example, aminoalkylphosphoramidates, aminoalkylphosphotriesters),those containing pendant moieties, such as, for example, proteins(including enzymes (for example, nucleases), toxins, antibodies, signalpeptides, poly-L-lysine, or the like), those with intercalators (forexample, acridine, psoralen, or the like), those containing chelates(of, for example, metals, radioactive metals, boron, oxidative metals,or the like), those containing alkylators, those with modified linkages(for example, alpha anomeric nucleic acids, or the like), as well asunmodified forms of the polynucleotide or oligonucleotide.

A “promoter” means a nucleic acid sequence sufficient to directtranscription of an operably linked nucleic acid molecule. A promotercan be used with or without other transcription control elements (forexample, enhancers) that are sufficient to render promoter-dependentgene expression controllable in a cell type-specific, tissue-specific,or temporal-specific manner, or that are inducible by external signalsor agents; such elements, may be within the 3′ region of a gene orwithin an intron. Desirably, a promoter is operably linked to a nucleicacid sequence, for example, a cDNA or a gene sequence, or an effectorRNA coding sequence, in such a way as to enable expression of thenucleic acid sequence, or a promoter is provided in an expressioncassette into which a selected nucleic acid sequence to be transcribedcan be conveniently inserted. A regulatory element such as promoteractive in a mammalian cells means a regulatory element configurable toresult in a level of expression of at least 1 transcript per cell in amammalian cell into which the regulatory element has been introduced.

The term “selectable marker” means a polynucleotide segment orexpression product thereof that allows one to select for or against amolecule or a cell that contains it, often under particular conditions.These markers can encode an activity, such as, but not limited to,production of RNA, peptide, or protein, or can provide a binding sitefor RNA, peptides, proteins, inorganic and organic compounds orcompositions. Examples of selectable markers include but are not limitedto: (1) DNA segments that encode products which provide resistanceagainst otherwise toxic compounds (e.g., antibiotics); (2) DNA segmentsthat encode products which are otherwise lacking in the recipient cell(e.g., tRNA genes, auxotrophic markers); (3) DNA segments that encodeproducts which suppress the activity of a gene product; (4) DNA segmentsthat encode products which can be readily identified (e.g., phenotypicmarkers such as beta-galactosidase, green fluorescent protein (GFP), andcell surface proteins); (5) DNA segments that bind products which areotherwise detrimental to cell survival and/or function; (6) DNA segmentsthat otherwise inhibit the activity of any of the DNA segments describedin Nos. 1-5 above (e.g., antisense oligonucleotides); (7) DNA segmentsthat bind products that modify a substrate (e.g. restrictionendonucleases); (8) DNA segments that can be used to isolate a desiredmolecule (e.g. specific protein binding sites); (9) DNA segments thatencode a specific nucleotide sequence which can be otherwisenon-functional (e.g., for PCR amplification of subpopulations ofmolecules); and/or (10) DNA segments, which when absent, directly orindirectly confer sensitivity to particular compounds.

Sequence identity can be determined by aligning sequences usingalgorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), using default gap parameters, or by inspection, and thebest alignment (i.e., resulting in the highest percentage of sequencesimilarity over a comparison window). Percentage of sequence identity iscalculated by comparing two optimally aligned sequences over a window ofcomparison, determining the number of positions at which the identicalresidues occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof matched and mismatched positions not counting gaps in the window ofcomparison (i.e., the window size), and multiplying the result by 100 toyield the percentage of sequence identity. Unless otherwise indicatedthe window of comparison between two sequences is defined by the entirelength of the shorter of the two sequences.

A “target nucleic acid” is a nucleic acid into which a transposon is tobe inserted. Such a target can be part of a chromosome, episome orvector.

An “integration target sequence” or “target sequence” or “target site”for a transposase is a site or sequence in a target DNA molecule intowhich a transposon can be inserted by a transposase. The piggyBactransposase from Trichoplusia ni inserts its transposon predominantlyinto the target sequence 5′-TTAA-3′. Other useable target sequences forpiggyBac transposons are 5′-CTAA-3′, 5′-TTAG-3′, 5′-ATAA-3′, 5′-TCAA-3′,5′-AGTT-3′, 5′-ATTA-3′, 5′-GTTA-3′, 5′-TTGA-3′, 5′-TTTA-3′, 5′-TTAC-3′,5′-ACTA-3′, 5′-AGGG-3′, 5′-CTAG-3′, 5′-GTAA-3′, 5′-AGGT-3′, 5′-ATCA-3′,5′-CTCC-3′, 5′-TAAA-3′, 5′-TCTC-3′, 5′-TGAA-3′, 5′-AAAT-3′, 5′-AATC-3′,5′-ACAA-3′, 5′-ACAT-3′, 5′-ACTC-3′, 5′-AGTG-3′, 5′-ATAG-3′, 5′-CAAA-3′,5′-CACA-3′, 5′-CATA-3′, 5′-CCAG-3′, 5′-CCCA-3′, 5′-CGTA-3′, 5′-CTGA-3′,5′-GTCC-3′, 5′-TAAG-3′, 5′-TCTA-3′, 5′-TGAG-3′, 5′-TGTT-3′, 5′-TTCA-3′,5′-TTCT-3′ and 5′-TTTT-3′ (Li et al., 2013. Proc. Natl. Acad. Sci vol.110, no. 6, E478-487). PiggyBac-like transposases transpose theirtransposons using a cut-and-paste mechanism, which results induplication of their 4 base pair target sequence on insertion into a DNAmolecule. The target sequence is thus found on each side of anintegrated piggyBac-like transposon.

The term “translation” refers to the process by which a polypeptide issynthesized by a ribosome ‘reading’ the sequence of a polynucleotide.

A ‘transposase’ is a polypeptide that catalyzes the excision of acorresponding transposon from a donor polynucleotide, for example avector, and (providing the transposase is not integration-deficient) thesubsequent integration of the transposon into a target nucleic acid. An“Amyelois transposase” means a transposase with at least 80%, 90, 95,96, 7, 98, 99 or 100% sequence identity to SEQ ID NO: 18, includinghyperactive variants of SEQ ID NO: 18, that are able to transposase acorresponding transposon. A hyperactive transposase is a transposasethat is more active than the naturally occurring transposase from whichit is derived, for excision activity or transposition activity or both.A hyperactive transposase is preferably at least 1.5-fold more active,or at least 2-fold more active, or at least 5-fold more active, or atleast 10-fold more active than the naturally occurring transposase fromwhich it is derived, e.g., 2-5 fold or 2-10 fold. A transposase may ormore not be fused to one or more additional domains such as a nuclearlocalization sequence or DNA binding protein.

The term “transposition” is used herein to mean the action of atransposase in excising a transposon from one polynucleotide and thenintegrating it, either into a different site in the same polynucleotide,or into a second polynucleotide.

The term “transposon” means a polynucleotide that can be excised from afirst polynucleotide, for instance, a vector, and be integrated into asecond position in the same polynucleotide, or into a secondpolynucleotide, for instance, the genomic or extrachromosomal DNA of acell, by the action of a corresponding trans-acting transposase. Atransposon comprises a first transposon end and a second transposon end,which are polynucleotide sequences recognized by and transposed by atransposase. A transposon usually further comprises a firstpolynucleotide sequence between the two transposon ends, such that thefirst polynucleotide sequence is transposed along with the twotransposon ends by the action of the transposase. This firstpolynucleotide in natural transposons frequently comprises an openreading frame encoding a corresponding transposase that recognizes andtransposes the transposon. Transposons of the present invention are“synthetic transposons” comprising a heterologous polynucleotidesequence which is transposable by virtue of its juxtaposition betweentwo transposon ends. Synthetic transposons may or may not furthercomprise flanking polynucleotide sequence(s) outside the transposonends, such as a sequence encoding a transposase, a vector sequence orsequence encoding a selectable marker.

The term “transposon end” means the cis-acting nucleotide sequences thatare sufficient for recognition by and transposition by a correspondingtransposase. Transposon ends of piggyBac-like transposons compriseperfect or imperfect repeats such that the respective repeats in the twotransposon ends are reverse complements of each other. These arereferred to as inverted terminal repeats (ITR) or terminal invertedrepeats (TIR). A transposon end may or may not include additionalsequence proximal to the ITR that promotes or augments transposition.

The term “vector” or “DNA vector” or “gene transfer vector” refers to apolynucleotide that is used to perform a “carrying” function for anotherpolynucleotide. For example, vectors are often used to allow apolynucleotide to be propagated within a living cell, or to allow apolynucleotide to be packaged for delivery into a cell, or to allow apolynucleotide to be integrated into the genomic DNA of a cell. A vectormay further comprise additional functional elements, for example it maycomprise a transposon.

5.2 Description

5.2.1 Genomic Integration

Expression of a gene from a heterologous polynucleotide in a eukaryotichost cell can be improved if the heterologous polynucleotide isintegrated into the genome of the host cell. Integration of apolynucleotide into the genome of a host cell also generally makes itstably heritable, by subjecting it to the same mechanisms that ensurethe replication and division of genomic DNA. Such stable heritability isdesirable for achieving good and consistent expression over long growthperiods. This is particularly important for cell therapies in whichcells are genetically modified and then placed into the body. It is alsoimportant for the manufacturing of biomolecules, particularly fortherapeutic applications where the stability of the host and consistencyof expression levels is also important for regulatory purposes. Cellswith gene transfer vectors, including transposon-based gene transfervectors, integrated into their genomes are thus an important embodimentof the invention.

Heterologous polynucleotides may be more efficiently integrated into atarget genome if they are part of a transposon (i.e., positioned betweentransposon ITRs), for example so that they may be integrated by atransposase A particular benefit of a transposon is that the entirepolynucleotide between the transposon ITRs is integrated. A transposoncomprising target sites flanking ITRs flanking a heterologouspolynucleotide integrates at a target site in a genome to result in thegenome containing the heterologous polynucleotide flanked by the ITRs,flanked by target sites. This is in contrast to random integration,where a polynucleotide introduced into a eukaryotic cell is oftenfragmented at random in the cell, and only parts of the polynucleotidebecome incorporated into the target genome, usually at a low frequency.The piggyBac transposon from the looper moth Trichoplusia ni has beenshown to be transposed by its transposase in cells from many organisms(see e.g. Keith et al (2008) BMC Molecular Biology 9:72 “Analysis of thepiggyBac transposase reveals a functional nuclear targeting signal inthe 94 c-terminal residues”). Heterologous polynucleotides incorporatedinto piggyBac-like transposons may be integrated into eukaryotic cellsincluding animal cells, fungal cells or plant cells. Preferred animalcells can be vertebrate or invertebrate. Preferred vertebrate cellsinclude cells from mammals including rodents such as rats, mice, andhamsters; ungulates, such as cows, goats or sheep; and swine. Preferredvertebrate cells also include cells from human tissues and human stemcells. Target cells types include hepatocytes, neural cells, musclecells, blood cells, embryonic stem cells, somatic stem cells,hematopoietic cells, embryos, zygotes, sperm cells (some of which areopen to be manipulated in an in vitro setting) and immune cellsincluding lymphocytes such as T cells, B cells and natural killer cells,T-helper cells, antigen-presenting cells, dendritic cells, neutrophilsand macrophages. Preferred cells can be pluripotent cells (cells whosedescendants can differentiate into several restricted cell types, suchas hematopoietic stem cells or other stem cells) or totipotent cells(i.e., a cell whose descendants can become any cell type in an organism,e.g., embryonic stem cells). Preferred culture cells are Chinese hamsterovary (CHO) cells or Human embryonic kidney (HEK293) cells. Preferredfungal cells are yeast cells including Saccharomyces cerevisiae andPichia pastoris. Preferred plant cells are algae, for example Chlorella,tobacco, maize and rice (Nishizawa-Yokoi et al (2014) Plant J. 77:454-63“Precise marker excision system using an animal derived piggyBactransposon in plants”).

Preferred gene transfer systems comprise a transposon in combinationwith a corresponding transposase protein that transposases thetransposon, or a nucleic acid that encodes the corresponding transposaseprotein and is expressible in the target cell. A preferred gene transfersystem comprises a synthetic Amyelois transposon and a correspondingAmyelois transposase.

A transposase protein can be introduced into a cell as a protein or as anucleic acid encoding the transposase, for example as a ribonucleicacid, including mRNA or any polynucleotide recognized by thetranslational machinery of a cell; as DNA, e.g. as extrachromosomal DNAincluding episomal DNA; as plasmid DNA, or as viral nucleic acid.Furthermore, the nucleic acid encoding the transposase protein can betransfected into a cell as a nucleic acid vector such as a plasmid, oras a gene expression vector, including a viral vector. The nucleic acidcan be circular or linear. mRNA encoding the transposase may be preparedusing DNA in which a gene encoding the transposase is operably linked toa heterologous promoter, such as the bacterial T7 promoter, which isactive in vitro. DNA encoding the transposase protein can be stablyinserted into the genome of the cell or into a vector for constitutiveor inducible expression. Where the transposase protein is transfectedinto the cell or inserted into the vector as DNA, the transposaseencoding sequence is preferably operably linked to a heterologouspromoter. There are a variety of promoters that could be used includingconstitutive promoters, cell-type specific promoters, organism-specificpromoters, tissue-specific promoters, inducible promoters, and the like.Where DNA encoding the transposase is operably linked to a promoter andtransfected into a target cell, the promoter should be operable in thetarget cell. For example if the target cell is a mammalian cell, thepromoter should be operable in a mammalian cell; if the target cell is ayeast cell, the promoter should be operable in a yeast cell; if thetarget cell is an insect cell, the promoter should be operable in aninsect cell; if the target cell is a human cell, the promoter should beoperable in a human cell; if the target cell is a human immune cell, thepromoter should be operable in a human immune cell. All DNA or RNAsequences encoding piggyBac-like transposase proteins are expresslycontemplated. Alternatively, the transposase may be introduced into thecell directly as protein, for example using cell-penetrating peptides(e.g. as described in Ramsey and Flynn (2015) Pharmacol. Ther. 154:78-86 “Cell-penetrating peptides transport therapeutics into cells”);using small molecules including salt plus propanebetaine (e.g. asdescribed in Astolfo et al (2015) Cell 161: 674-690); or electroporation(e.g. as described in Morgan and Day (1995) Methods in Molecular Biology48: 63-71 “The introduction of proteins into mammalian cells byelectroporation”).

It is possible to insert the transposon into DNA of a cell throughnon-homologous recombination through a variety of reproduciblemechanisms, and even without the activity of a transposase. Thetransposons described herein can be used for gene transfer regardless ofthe mechanisms by which the genes are transferred.

5.2.5 Gene Transfer Systems

Gene transfer systems comprise a polynucleotide to be transferred to ahost cell. Preferably the polynucleotide comprises an Amyeloistransposon and wherein the polynucleotide is to be integrated into thegenome of a target cell.

When there are multiple components of a gene transfer system, forexample the one or more polynucleotides comprising genes for expressionin the target cell and optionally comprising transposon ends, and atransposase (which may be provided either as a protein or encoded by anucleic acid), these components can be transfected into a cell at thesame time, or sequentially. For example, a transposase protein or itsencoding nucleic acid may be transfected into a cell prior to,simultaneously with or subsequent to transfection of a correspondingtransposon. Additionally, administration of either component of the genetransfer system may occur repeatedly, for example, by administering atleast two doses of this component.

Any of the transposase proteins described herein may be encoded bypolynucleotides including RNA or DNA. Similarly, the nucleic acidencoding the transposase protein or the transposon of this invention canbe transfected into the cell as a linear fragment or as a circularizedfragment, either as a plasmid or as recombinant viral DNA.

An Amyelois transposase may be provided as a DNA molecule expressible inthe target cell. The sequence encoding the Amyelois transposase shouldbe operably linked to heterologous sequences that enable expression ofthe transposase in the target cell. A sequence encoding the Amyeloistransposase may be operably linked to a heterologous promoter that isactive in the target cell. For example, if the target cell is amammalian cell, then the promoter should be active in a mammalian cell.If the target is a vertebrate cell, the promoter should be active in avertebrate cell. If the target cell is a plant cell, the promoter shouldbe active in a plant cell. If the promoter is an insect cell, thepromoter should be active in an insect cell. The sequence encoding theAmyelois transposase may also be operably linked to other sequenceelements required for expression in the target cell, for examplepolyadenylation sequences, terminator sequences etc.

An Amyelois transposase may be provided as an mRNA expressible in thetarget cell. mRNA is preferably prepared in an in vitro transcriptionreaction. For in vitro transcription, a sequence encoding the Amyeloistransposase is operably linked to a promoter that is active in an invitro transcription reaction. Exemplary promoters active in an in vitrotranscription reaction include a T7 promoter (5′-TAATACGACTCACTATAG-3′)which enables transcription by T7 RNA polymerase, a T3 promoter(5′-AATTAACCCTCACTAAAG-3′) which enables transcription by T3 RNApolymerase and an SP6 promoter (5′-ATTTAGGTGACACTATAG-3′) which enablestranscription by SP6 RNA polymerase. Variants of these promoters andother promoters that can be used for in vitro transcription may also beoperably linked to a sequence encoding an Amyelois transposase.

If the Amyelois transposase is provided as a polynucleotide (either DNAor mRNA) encoding the transposase, then it is advantageous to improvethe expressibility of the transposase in the target cell. It istherefore advantageous to use a sequence other than a naturallyoccurring sequence to encode the transposase, in other words, to usecodon-preferences of the cell type in which expression is to beperformed. For example, if the target cell is a mammalian cell, then thecodons should be biased toward the preferences seen in a mammalian cell.If the target is a vertebrate cell, then the codons should be biasedtoward the preferences seen in the particular vertebrate cell. If thetarget cell is a plant cell, then the codons should be biased toward thepreferences seen in a in a plant cell. If the promoter is an insectcell, then the codons should be biased toward the preferences seen in aninsect cell.

Preferable RNA molecules include those with appropriate cap structuresto enhance translation in a eukaryotic cell, polyadenylic acid and other3′ sequences that enhance mRNA stability in a eukaryotic cell andoptionally substitutions to reduce toxicity effects on the cell, forexample substitution of uridine with pseudouridine, and substitution ofcytosine with 5-methyl cytosine. mRNA encoding the Amyelois transposasemay be prepared such that it has a 5′-cap structure to improveexpression in a target cell. Exemplary cap structures are a cap analog(G(5′)ppp(5′)G), an anti-reverse cap analog (3′-O-Me-m⁷G(5′)ppp(5′)G, aclean cap (m7G(5′)ppp(5)(2′OMeA)pG) an mCap On7G(5′)ppp(5′)G). mRNAencoding the Amyelois transposase may be prepared such that some basesare partially or fully substituted, for example uridine may besubstituted with pseudo-uridine, cytosine may be substituted with5-methyl-cytosine. Any combinations of these caps and substitutions maybe made.

The components of the gene transfer system may be transfected into oneor more cells by techniques such as particle bombardment,electroporation, microinjection, combining the components withlipid-containing vesicles, such as cationic lipid vesicles, DNAcondensing reagents (example, calcium phosphate, polylysine orpolyethyleneimine), and inserting the components (that is the nucleicacids thereof into a viral vector and contacting the viral vector withthe cell. Where a viral vector is used, the viral vector can include anyof a variety of viral vectors known in the art including viral vectorsselected from the group consisting of a retroviral vector, an adenovirusvector or an adeno-associated viral vector. The gene transfer system maybe formulated in a suitable manner as known in the art, or as apharmaceutical composition or kit.

5.2.3 Sequence Elements in Gene Transfer Systems

Expression of genes from a gene transfer polynucleotide such as apiggyBac-like transposon, including an Amyelois transposon, integratedinto a host cell genome is often strongly influenced by the chromatinenvironment into which it integrates. Polynucleotides that areintegrated into euchromatin have higher levels of expression than thosethat are either integrated into heterochromatin, or which becomesilenced following their integration. Silencing of a heterologouspolynucleotide may be reduced if it comprises a chromatin controlelement. It is thus advantageous for gene transfer polynucleotides(including any of the transposons described herein) to comprisechromatin control elements such as sequences that prevent the spread ofheterochromatin (insulators). Advantageous gene transfer polynucleotidesincluding an Amyelois transposon comprise an insulator sequence that isat least 95% identical to a sequence selected from one of SEQ ID NOS:435-441, they may also comprise ubiquitously acting chromatin openingelements (UCOEs) or stabilizing and anti-repressor elements (STARs), toincrease long-term stable expression from the integrated gene transferpolynucleotide. Advantageous gene transfer polynucleotides may furthercomprise a matrix attachment region for example a sequence that is atleast 95% identical to a sequence selected from one of SEQ ID NOS:442-452.

In some cases, it is advantageous for a gene transfer polynucleotide tocomprise two insulators, one on each side of the heterologouspolynucleotide that contains the sequence(s) to be expressed, and withinthe transposon ITRs. The insulators may be the same, or they may bedifferent. Particularly advantageous gene transfer polynucleotidescomprise an insulator sequence that is at least 95% identical to asequence selected from one of SEQ ID NO: 440 or SEQ ID NO: 441 and aninsulator sequence that is at least 95% identical to a sequence selectedfrom one of SEQ ID NOS: 435-439. Insulators also shield expressioncontrol elements from one another. For example, when a gene transferpolynucleotide comprises genes encoding two open reading frames, eachoperably linked to a different promoter, one promoter may reduceexpression from the other in a phenomenon known as transcriptionalinterference. Interposing an insulator sequence that is at least 95%identical to a sequence selected from one of SEQ ID NOS: 435-441 betweenthe two transcriptional units can reduce this interference, increasingexpression from one or both promoters.

Preferred gene transfer vectors comprise expression elements capable ofdriving high levels of gene expression. In eukaryotic cells, geneexpression is regulated by several different classes of elements,including enhancers, promoters, introns, RNA export elements,polyadenylation sequences and transcriptional terminators.

Advantageous gene transfer polynucleotides for the transfer of genes forexpression into eukaryotic cells comprise an enhancer operably linked toa heterologous gene. Advantageous gene transfer polynucleotides for thetransfer of genes for expression into mammalian cells comprise anenhancer from immediate early genes 1, 2 or 3 of cytomegalovirus (CMV)from either human, primate or rodent cells (for example sequences atleast 95% identical to any of SEQ ID NOS: 453-471), an enhancer from theadenoviral major late protein enhancer (for example sequences at least95% identical to SEQ ID NO: 472), or an enhancer from SV40 (for examplesequences at least 95% identical to SEQ ID NO: 473), operably linked toa heterologous gene.

Advantageous gene transfer polynucleotides for the transfer of genes forexpression into eukaryotic cells comprise a promoter operably linked toa heterologous gene. Advantageous gene transfer polynucleotides for thetransfer of genes for expression into mammalian cells comprise an EF1apromoter from any mammalian or avian species including human, rat, mice,chicken and Chinese hamster, (for example any of SEQ ID NOS: 474-495); apromoter from the immediate early genes 1, 2 or 3 of cytomegalovirus(CMV) from either human, primate or rodent cells (for example any of SEQID NOS: 496-506); a promoter for eukaryotic elongation factor 2 (EEF2)from any mammalian or avian species including human, rat, mice, chickenand Chinese hamster, (for example any of SEQ ID NOS: 507-517); a GAPDHpromoter from any mammalian or yeast species (for example any of SEQ IDNOS: 528-544), an actin promoter from any mammalian or avian speciesincluding human, rat, mice, chicken and Chinese hamster (for example anyof SEQ ID NOS: 518-527); a PGK promoter from any mammalian or avianspecies including human, rat, mice, chicken and Chinese hamster (forexample any of SEQ ID NOS: 545-551), or a ubiquitin promoter (forexample SEQ ID NO: 552), operably linked to a heterologous gene. Thepromoter may be operably linked to i) a heterologous open reading frame;ii) a nucleic acid encoding a selectable marker; iii) a nucleic acidencoding a counter-selectable marker; iii) a nucleic acid encoding aregulatory protein; iv) a nucleic acid encoding an inhibitory RNA.

Advantageous gene transfer polynucleotides for the transfer of genes forexpression into eukaryotic cells comprise an intron within aheterologous polynucleotide spliceable in a target cell. Advantageousgene transfer polynucleotides for the transfer of genes for expressioninto mammalian cells comprise an intron from immediate early genes 1, 2or 3 of cytomegalovirus (CMV) from either human, primate or rodent cells(for example sequences at least 95% identical to any of SEQ ID NOS:561-571), an intron from EF1a from any mammalian or avian speciesincluding human, rat, mice, chicken and Chinese hamster, (for examplesequences at least 95% identical to any of SEQ ID NOS: 581-593), anintron from EEF2 from any mammalian or avian species including human,rat, mice, chicken and Chinese hamster, (for example sequences at least95% identical to any of SEQ ID NOS: 613-620); an intron from actin fromany mammalian or avian species including human, rat, mice, chicken andChinese hamster (for example sequences at least 95% identical to any ofSEQ ID NOS: 594-607), a GAPDH intron from any mammalian or avian speciesincluding human, rat, mice, chicken and Chinese hamster (for examplesequences at least 95% identical to any of SEQ ID NOS: 608-610); anintron comprising the adenoviral major late protein enhancer for examplesequences at least 95% identical to SEQ ID NO: 611-612) or ahybrid/synthetic intron (for example sequences at least 95% identical toany of SEQ ID NOS: 572-580) within a heterologous polynucleotide.

Advantageous gene transfer polynucleotides for the transfer of genes forexpression into eukaryotic cells comprise an enhancer and promoter,operably linked to a heterologous coding sequence. Such gene transferpolynucleotides may comprise combinations of enhancers and promoters inwhich an enhancer from one gene is combined with a promoter from adifferent gene, that is the enhancer is heterologous to the promoter.For example, for the transfer of genes for expression into mammaliancells, an immediate early CMV enhancer from rodent or human or primate(such as a sequence selected from SEQ ID NOS: 453-471) is advantageouslyfollowed by a promoter from an EF1a gene (such as a sequence selectedfrom SEQ ID NOS: 474-495), or a promoter from a heterologous CMV gene(such as a sequence selected from SEQ ID NOS: 496-506), or a promoterfrom an EEF2 gene (such as a sequence selected from SEQ ID NOS:507-517), or a promoter from an actin gene (such as a sequence selectedfrom SEQ ID NOS: 518-527), or a promoter from a GAPDH gene (such as asequence selected from SEQ ID NOS: 528-544) operably linked to aheterologous sequence.

Advantageous gene transfer polynucleotides for the transfer of genes forexpression into eukaryotic cells comprise an operably linked promoterand an intron, operably linked to a heterologous open reading frame.Such gene transfer polynucleotides may comprise combinations ofpromoters and introns in which a promoter from one gene is combined withan intron from a different gene, that is the intron is heterologous tothe promoter. For example, for the transfer of genes for expression intomammalian cells, an immediate early CMV promoter from rodent or human orprimate (such as a sequence selected from SEQ ID NOS: 496-506) isadvantageously followed by an intron from an EF1a gene (such as asequence that is at least 95% identical to a sequence selected from SEQID NOS: 581-593) or an intron from an EEF2 gene (such as a sequence thatis at least 95% identical to a sequence selected from SEQ ID NOS:613-620), or an intron from an actin gene (such as a sequence that is atleast 95% identical to a sequence selected from SEQ ID NOS: 594-607)operably linked to a heterologous sequence.

Advantageous gene transfer polynucleotides for the transfer of genes forexpression into eukaryotic cells, comprise composite transcriptionalinitiation regulatory elements comprising promoters that are operablylinked to enhancers and/or introns, and the composite transcriptionalinitiation regulatory element is operably linked to a heterologoussequence. Examples of advantageous composite transcriptional initiationregulatory elements that may be operably linked to a heterologoussequence in gene transfer polynucleotides for the transfer of genes forexpression into mammalian cells are sequences selected from SEQ ID NO:622-714.

Expression of two open reading frames from a single polynucleotide canbe accomplished by operably linking the expression of each open readingframe to a separate promoter, each of which may optionally be operablylinked to enhancers and introns as described above. This is particularlyuseful when expressing two polypeptides that need to interact atspecific molar ratios, such as chains of an antibody or chains of abispecific antibody, or a receptor and its ligand. It is oftenadvantageous to prevent transcriptional promoter interference by placinga genetic insulator between the two open reading frames, for example tothe 3′ of the polyadenylation sequence operably linked to the first openreading frame and to the 5′ of the promoter operably linked to thesecond open reading frame encoding the second polypeptide.Transcriptional promoter interference may also be prevented byeffectively terminating transcription of the first gene. In manyeukaryotic cells the use of strong polyA signal sequences between twoopen reading frames will reduce transcriptional promote interference.Examples of polyA signal sequences that can be used to effectivelyterminate transcription are given as SEQ ID NOs: 715-744. Advantageousgene transfer polynucleotides comprise a sequence that is at least 95%identical to a sequence selected from SEQ ID NOs: 715-744 operablylinked to a heterologous open reading frame. Advantageous compositeregulatory elements for the termination of transcription of a first geneand the initiation of transcription of a second gene include sequencesgiven as SEQ ID NOs: 745-928. Particularly advantageous gene transferpolynucleotides for the transfer of a first and a second open readingframe for co-expression into mammalian cells comprise a sequence atleast 90% identical or at least 95% identical or at least 99% identicalto or 100% identical to a sequence selected from SEQ ID NOS: 745-928,separating two heterologous open reading frames.

5.2.4 Selection of Target Cells Comprising Gene Transfer Polynucleotides

A target cell whose genome comprises a stably integrated transferpolynucleotide may be identified, if the gene transfer polynucleotidecomprises an open reading frame encoding a selectable marker, byexposing the target cells to conditions that favor cells expressing theselectable marker (“selection conditions”). It is advantageous for agene transfer polynucleotide to comprise an open reading frame encodinga selectable marker such as an enzyme that confers resistance toantibiotics such as neomycin (resistance conferred by an aminoglycoside3′-phosphotransferase e.g. a sequence selected from SEQ ID NOs:262-265), puromycin (resistance conferred by puromycin acetyltransferasee.g. a sequence selected from SEQ ID NOs: 268-270), blasticidin(resistance conferred by a blasticidin acetyltransferase and ablasticidin deaminase e.g. SEQ ID NO: 272), hygromycin B (resistanceconferred by hygromycin B phosphotransferase e.g. a sequence selectedfrom SEQ ID NOs: 266-267) and zeocin (resistance conferred by a bindingprotein encoded by the ble gene, for example SEQ ID NO: 259). Otherselectable markers include those that are fluorescent (such as openreading frames encoding GFP, RFP etc.) and can therefore be selected forexample using flow cytometry. Other selectable markers include openreading frames encoding transmembrane proteins that are able to bind toa second molecule (protein or small molecule) that can be fluorescentlylabelled so that the presence of the transmembrane protein can beselected for example using flow cytometry.

A gene transfer polynucleotide may comprise a selectable marker openreading frame encoding glutamine synthetase (GS, for example a sequenceselected from SEQ ID NOS: 274-278) which allows selection via glutaminemetabolism. Glutamine synthase is the enzyme responsible for thebiosynthesis of glutamine from glutamate and ammonia, it is a crucialcomponent of the only pathway for glutamine formation in a mammaliancell. In the absence of glutamine in the growth medium, the GS enzyme isessential for the survival of mammalian cells in culture. Some celllines, for example mouse myeloma cells do not express sufficient GSenzyme to survive without added glutamine. In these cells a transfectedGS open reading frame can function as a selectable marker by permittinggrowth in a glutamine-free medium. In other cell lines, for exampleChinese hamster ovary (CHO) cells express sufficient GS enzyme tosurvive without exogenously added glutamine. These cell lines can bemanipulated by genome editing techniques including CRISPR/Cas9 to reduceor eliminate the activity of the GS enzyme. In all of these cases, GSinhibitors such as methionine sulphoximine (MSX) can be used to inhibita cell's endogenous GS activity. Selection protocols include introducinga gene transfer polynucleotide comprising sequences encoding a firstpolypeptide and a glutamine synthase selectable marker, and thentreating the cell with inhibitors of glutamine synthase such asmethionine sulphoximine. The higher the levels of methioninesulphoximine that are used, the higher the level of glutamine synthaseexpression is required to allow the cell to synthesize sufficientglutamine to survive. Some of these cells will also show an increasedexpression of the first polypeptide.

Preferably the GS open reading frame is operably linked to a weakpromoter or other sequence elements that attenuate expression asdescribed herein, such that high levels of expression can only occur ifmany copies of the gene transfer polynucleotide are present, or if theyare integrated in a position in the genome where high levels ofexpression occur. In such cases it may be unnecessary to use theinhibitor methionine sulphoximine: simply synthesizing sufficientglutamine for cell survival may provide a sufficiently stringentselection if expression of the glutamine synthetase is attenuated.

A gene transfer polynucleotide may comprise a selectable marker openreading frame encoding dihydrofolate reductase (DHFR, for example asequence selected from SEQ ID NO: 260-261) which is required forcatalyzing the reduction of 5,6-dihydrofolate (DHF) to5,6,7,8-tetrahydrofolate (THF). Some cell lines do not expresssufficient DHFR to survive without added hypoxanthine and thymidine(HT). In these cells a transfected DHFR open reading frame can functionas a selectable marker by permitting growth in a hypoxanthine andthymidine-free medium. DHFR-deficient cell lines, for example Chinesehamster ovary (CHO) cells can be produced by genome editing techniquesincluding CRISPR/Cas9 to reduce or eliminate the activity of theendogenous DHRF enzyme. DHFR confers resistance to methotrexate (MTX).DHFR can be inhibited by higher levels of methotrexate. Selectionprotocols include introducing a construct comprising sequences encodinga first polypeptide and a DHFR selectable marker into a cell with orwithout a functional endogenous DHFR gene, and then treating the cellwith inhibitors of DHFR such as methotrexate. The higher the levels ofmethotrexate that are used, the higher the level of DHFR expression isrequired to allow the cell to synthesize sufficient DHFR to survive.Some of these cells will also show an increased expression of the firstpolypeptide. Preferably the DHFR open reading frame is operably linkedto a weak promoter or other sequence elements that attenuate expressionas described above, such that high levels of expression can only occurif many copies of the gene transfer polynucleotide are present, or ifthey are integrated in a position in the genome where high levels ofexpression occur.

High levels of expression may be obtained from genes encoded on genetransfer polynucleotides that are integrated at regions of the genomethat are highly transcriptionally active, or that are integrated intothe genome in multiple copies, or that are present extrachromosomally inmultiple copies. It is often advantageous to operably link the openreading frame encoding the selectable marker to expression controlelements that result in low levels of expression of the selectablepolypeptide from the gene transfer polynucleotide and/or to useconditions that provide more stringent selection. Under theseconditions, for the expression cell to produce sufficient levels of theselectable polypeptide encoded on the gene transfer polynucleotide tosurvive the selection conditions, the gene transfer polynucleotide caneither be present in a favorable location in the cell's genome for highlevels of expression, or a sufficiently high number of copies of thegene transfer polynucleotide can be present, such that these factorscompensate for the low levels of expression achievable because of theexpression control elements.

Genomic integration of transposons in which a selectable marker isoperably linked to regulatory elements that only weakly express themarker usually requires that the transposon be inserted into the targetgenome by a transposase, see for example Section 6.1.3. By operablylinking the selectable marker to elements that result in weakexpression, cells are selected which either incorporate multiple copiesof the transposon, or in which the transposon is integrated at afavorable genomic location for high expression. Using a gene transfersystem that comprises a transposon and a corresponding transposaseincreases the likelihood that cells will be produced with multiplecopies of the transposon, or in which the transposon is integrated at afavorable genomic location for high expression. Gene transfer systemscomprising a transposon and a corresponding transposase are thusparticularly advantageous when the transposon comprises a selectablemarker operably linked to a weak promoter.

A nucleic acid to be expressed as an RNA or protein and a selectablemarker may be included on the same gene transfer polynucleotide, butoperably linked to different promoters. In this case low expressionlevels of the selectable marker may be achieved by using a weakly activeconstitutive promoter such as the phosphoglycerokinase (PGK) promoter(such as a promoter selected from SEQ ID NOS: 545-551), the HerpesSimplex Virus thymidine kinase (HSV-TK) promoter (e.g. a sequenceselected from SEQ ID NOs: 553-554), the MC1 promoter (for example SEQ IDNO: 555), a ubiquitin promoter (for example a sequence selected from SEQID NO: 552). Other weakly active promoters may be deliberatelyconstructed, for example a promoter attenuated by truncation, such as atruncated SV40 promoter (for example a sequence selected from SEQ ID NO:556-557), a truncated HSV-TK promoter (for example SEQ ID NO: 553), or apromoter attenuated by insertion of a 5′UTR unfavorable for expression(for example a sequence selected from SEQ ID NOS: 559-560) between apromoter and the open reading frame encoding the selectable polypeptide.Particularly advantageous gene transfer polynucleotides comprise apromoter sequence selected from SEQ ID NOS: 545-558, operably linked toan open reading frame encoding a selectable marker.

Expression levels of a selectable marker may also be advantageouslyreduced by other mechanisms such as the insertion of the SV40 small tantigen intron after the open reading frame for the selectable marker.The SV40 small t intron accepts aberrant 5′ splice sites, which can leadto deletions within the preceding open reading frame in a fraction ofthe spliced mRNAs, thereby reducing expression of the selectable marker.Particularly advantageous gene transfer polynucleotides comprise intronSEQ ID NO: 621, operably linked to an open reading frame encoding aselectable marker. For this mechanism of attenuation to be effective, itis preferable for the open reading frame encoding the selectable markerto comprise an intron donor within its coding region. DNA sequences SEQID NOs: 279-282 are exemplary nucleic acid sequences that encodeglutamine synthetase sequences with SEQ ID NO: 274-277 respectively.Each of these nucleic acid sequences comprises an intron donor, andwhich may be operably linked to the SV40 small t antigen intron byplacing the intron into the 3′ UTR of the glutamine synthetase openreading frame. Sequence SEQ ID NO: 271 is an exemplary nucleic acidsequence encoding puromycin acetyl transferase SEQ ID NO: 270, whichcomprises an intron donor, and which may be operably linked to the SV40small t antigen intron by placing the intron into the 3′ UTR of thepuromycin open reading frame. Advantageous gene transfer polynucleotidescomprise a sequence at least 90% identical or at least 95% identical orat least 99% identical to, or 100% identical to a sequence selected fromone of SEQ ID NO: 279-282 or 271, operably linked to SEQ ID NO: 621.

Expression levels of a selectable marker may also be advantageouslyreduced by other mechanisms such as insertion of an inhibitory 5′-UTRwithin the transcript, for example SEQ ID NO: 559-560. Particularlyadvantageous gene transfer polynucleotides comprise a promoter operablylinked to an open reading frame encoding a selectable marker, wherein asequence that is at least 90% identical or at least 95% identical or atleast 99% identical to, or 100% identical to SEQ ID NO: 559-560 isinterposed between the promoter and the selectable marker.

Exemplary nucleic acid sequences comprising the glutamine synthetasecoding sequence operably linked to regulatory sequences expressible inmammalian cells include SEQ ID NOs: 300-370 or 432-434. A gene transferpolynucleotide comprising a sequence selected from SEQ ID NOs: 300-370or 432-434, upon integration into the genome of a target cell, expressesglutamine synthetase, thereby helping a cell to grow in the absence ofadded glutamine or in the presence of MSX. Regulatory elements in thesesequences have been balanced to produce low levels of expression ofglutamine synthetase, providing a selective advantage for target cellswhose genome comprises either multiple copies of the gene transferpolynucleotide, or for target calls whose genome comprises copies of thegene transfer polynucleotide in regions of the genome that are favorablefor expression of encoded genes. Advantageous gene transferpolynucleotides comprise a sequence selected from SEQ ID NOs: 300-370 or432-434, they may further comprise a left transposon end and a righttransposon end.

Exemplary nucleic acid sequences comprising theblasticidin-S-transferase coding sequence operably linked to regulatorysequences expressible in mammalian cells include SEQ ID NOs: 371-377. Agene transfer polynucleotide comprising a sequence selected from SEQ IDNOs: 371-377, upon integration into the genome of a target cell,expresses blasticidin-S-transferase, thereby helping a cell to grow inthe presence of added blasticidin. Regulatory elements in thesesequences have been balanced to produce low levels of expression ofblasticidin-S-transferase, providing a selective advantage for targetcells whose genome comprises either multiple copies of the gene transferpolynucleotide, or for target calls whose genome comprises copies of thegene transfer polynucleotide in regions of the genome that are favorablefor expression of encoded genes. Advantageous gene transferpolynucleotides comprise a sequence selected from SEQ ID NOs: 371-377,they may further comprise a left transposon end and a right transposonend.

Exemplary nucleic acid sequences comprising the hygromycin Bphosphotransferase coding sequence operably linked to regulatorysequences expressible in mammalian cells include SEQ ID NOs: 378-379. Agene transfer polynucleotide comprising a sequence selected from SEQ IDNOs: 378-379, upon integration into the genome of a target cell,expresses hygromycin B phosphotransferase, thereby helping a cell togrow in the presence of added hygromycin. Regulatory elements in thesesequences have been balanced to produce low levels of expression ofhygromycin B phosphotransferase, providing a selective advantage fortarget cells whose genome comprises either multiple copies of the genetransfer polynucleotide, or for target calls whose genome comprisescopies of the gene transfer polynucleotide in regions of the genome thatare favorable for expression of encoded genes. Advantageous genetransfer polynucleotides comprise a sequence selected from SEQ ID NOs:378-379, they may further comprise a left transposon end and a righttransposon end.

Exemplary nucleic acid sequences comprising the aminoglycoside3′-phosphotransferase coding sequence operably linked to regulatorysequences expressible in mammalian cells include SEQ ID NOs: 380-382 or408-409. A gene transfer polynucleotide comprising a sequence selectedfrom SEQ ID NOs: 380-382 or 408-409, upon integration into the genome ofa target cell, expresses aminoglycoside 3′-phosphotransferase, therebyhelping a cell to grow in the presence of added neomycin. Regulatoryelements in these sequences have been balanced to produce low levels ofexpression of aminoglycoside 3′-phosphotransferase, providing aselective advantage for target cells whose genome comprises eithermultiple copies of the gene transfer polynucleotide, or for target callswhose genome comprises copies of the gene transfer polynucleotide inregions of the genome that are favorable for expression of encodedgenes. Advantageous gene transfer polynucleotides comprise a sequenceselected from SEQ ID NOs: 380-382 or 408-409, they may further comprisea left transposon end and a right transposon end.

Exemplary nucleic acid sequences comprising the puromycinacetyltransferase coding sequence operably linked to regulatorysequences expressible in mammalian cells include SEQ ID NOs: 383-402 or410-434. A gene transfer polynucleotide comprising a sequence selectedfrom SEQ ID NOs: 383-402 or 410-434, upon integration into the genome ofa target cell, expresses puromycin acetyltransferase, thereby helping acell to grow in the presence of added puromycin. Regulatory elements inthese sequences have been balanced to produce low levels of expressionof puromycin acetyltransferase, providing a selective advantage fortarget cells whose genome comprises either multiple copies of the genetransfer polynucleotide, or for target calls whose genome comprisescopies of the gene transfer polynucleotide in regions of the genome thatare favorable for expression of encoded genes. Advantageous genetransfer polynucleotides comprise a sequence selected from SEQ ID NOs:383-402 or 410-434, they may further comprise a left transposon end anda right transposon end.

Exemplary nucleic acid sequences comprising the ble gene coding sequenceoperably linked to regulatory sequences expressible in mammalian cellsinclude SEQ ID NOs: 403-407. A gene transfer polynucleotide comprising asequence selected from SEQ ID NOs: 403-407, upon integration into thegenome of a target cell, expresses the ble gene, thereby helping a cellto grow in the presence of added zeocin. Regulatory elements in thesesequences have been balanced to produce low levels of expression of blegene product, providing a selective advantage for target cells whosegenome comprises either multiple copies of the gene transferpolynucleotide, or for target calls whose genome comprises copies of thegene transfer polynucleotide in regions of the genome that are favorablefor expression of encoded genes. Advantageous gene transferpolynucleotides comprise a sequence selected from SEQ ID NOs: 403-407,they may further comprise a left transposon end and a right transposonend.

Exemplary nucleic acid sequences comprising the dihydrofolate reductasecoding sequence operably linked to regulatory sequences expressible inmammalian cells include SEQ ID NOs: 283-299 or 408-431. A gene transferpolynucleotide comprising a sequence selected from SEQ ID NOs: 283-299or 408-431, upon integration into the genome of a target cell, expressesdihydrofolate reductase, thereby helping a cell to grow in the absenceof added hypoxanthine and thymidine or in the presence of MTX.Regulatory elements in these sequences have been balanced to produce lowlevels of expression of dihydrofolate reductase, providing a selectiveadvantage for target cells whose genome comprises either multiple copiesof the gene transfer polynucleotide, or for target calls whose genomecomprises copies of the gene transfer polynucleotide in regions of thegenome that are favorable for expression of encoded genes. Advantageousgene transfer polynucleotides comprise a sequence selected from SEQ IDNOs: 283-299 or 408-431, they may further comprise a left transposon endand a right transposon end.

The use of transposons and transposases in conjunction with weaklyexpressed selectable markers has several advantages over non-transposonconstructs. One is that linkage between expression of the firstpolypeptide and the selectable marker is better for transposons, becausea transposase integrates the entire sequence that lies between the twotransposon ends into the genome. In contrast when heterologous DNA isintroduced into the nucleus of a eukaryotic cell, for example amammalian cell, it is gradually broken into random fragments which mayeither be integrated into the cell's genome, or degraded. Thus, if agene transfer polynucleotide comprising sequences that encode a firstpolypeptide and a selectable marker is introduced into a population ofcells, some cells will integrate the sequences encoding the selectablemarker but not those encoding the first polypeptide, and vice versa.Selection of cells expressing high levels of selectable marker is thusonly somewhat correlated with cells that also express high levels of thefirst polypeptide. In contrast, because the transposase integrates allof the sequences between the transposon ends, cells expressing highlevels of selectable marker are highly likely to also express highlevels of the first polypeptide.

A second advantage of transposons and transposases is that they are muchmore efficient at integrating DNA sequences into the genome. A muchhigher fraction of the cell population is therefore likely to integrateone or more copies of the gene transfer polynucleotide into theirgenomes, so there will be a correspondingly higher likelihood of goodstable expression of both the selectable marker and the firstpolypeptide.

A third advantage of piggyBac-like transposons and transposases is thatpiggyBac-like transposases are biased toward inserting theircorresponding transposons into transcriptionally active chromatin. Eachcell is therefore likely to integrate the gene transfer polynucleotideinto a region of the genome from which genes are well expressed, sothere will be a correspondingly higher likelihood of good stableexpression of both the selectable marker and the first polypeptide.

5.2.5 a Novel PiggyBac-Like Transposase

Natural DNA transposons undergo a ‘cut and paste’ system of replicationin which the transposon is excised from a first DNA molecule andinserted into a second DNA molecule. DNA transposons are characterizedby inverted terminal repeats (ITRs) and are mobilized by anelement-encoded transposase. The piggyBac transposon/transposase systemis particularly useful because of the precision with which thetransposon is integrated and excised (see for example “Fraser, M. J.(2001) The TTAA-Specific Family of Transposable Elements:Identification, Functional Characterization, and Utility forTransformation of Insects. Insect Transgenesis: Methods andApplications. A. M. Handler and A. A. James. Boca Raton, Fla., CRCPress: 249-268”; and “US 20070204356 A1: PiggyBac constructs invertebrates” and references therein).

Many sequences with sequence similarity to the piggyBac transposase fromTrichoplusia ni have been found in the genomes of phylogeneticallydistinct species from fungi to mammals, but very few have been shown topossess transposase activity (see for example Wu M, et al (2011)Genetica 139:149-54. “Cloning and characterization of piggyBac-likeelements in lepidopteran insects”, and references therein).

Two properties of transposases that are of particular interest forgenomic modifications are their ability to integrate a polynucleotideinto a target genome, and their ability to precisely excise apolynucleotide from a target genome. Both of these properties can bemeasured with a suitable system.

A system for measuring the first step of transposition, which isexcision of a transposon from a first polynucleotide, comprises thefollowing components: (i) A first polynucleotide encoding a firstselectable marker operably linked to sequences that cause it to beexpressed in a selection host and (ii) A transposon comprisingtransposon ends recognized by a transposase. The transposon is presentin, and interrupts the coding sequence of, the first selectable marker,such that the first selectable marker is not active. The transposon isplaced in the first selectable marker such that precise excision of thefirst transposon causes the first selectable marker to be reconstituted.If an active transposase that can excise the first transposon isintroduced into a host cell which comprises the first polynucleotide,the host cell will express the active first selectable marker. Theactivity of the transposase in excising the transposon can be measuredas the frequency with which the host cells become able to grow underconditions that require the first selectable marker to be active.

If the transposon comprises a second selectable marker, operably linkedto sequences that make the second selectable marker expressible in theselection host, transposition of the second selectable marker into thegenome of the host cell will yield a genome comprising active first andsecond selectable markers. The activity of the transposase intransposing the transposon into a second genomic location can bemeasured as the frequency with which the host cells become able to growunder conditions that require the first and second selectable markers tobe active. In contrast, if the first selectable marker is present, butthe second is not, then this indicates that the transposon was excisedfrom the first polynucleotide but was not subsequently transposed into asecond polynucleotide. The selectable markers may, for example, be openreading frames encoding an antibiotic resistance protein, or anauxotrophic marker, or any other selectable marker.

We used such a system to test putative transposase/transposoncombinations for activity, as described in Section 6.1. We usedcomputational methods to search publicly available sequenced genomes foropen reading frames with homology to known active piggyBac-liketransposases. We selected transposase sequences that appeared to possessthe DDDE motif characteristic of active piggyBac-like transposases andsearched the DNA sequences flanking these putative transposases forinverted repeat sequences adjacent to a 5′-TTAA-3′ target sequence.Amongst those that we identified were putative transposons with intacttransposases from: Spodoptera litura (Genbank accession numberMTZO01002002.1, protein accession number XP_022823959) with an openreading frame encoding a putative transposase with SEQ ID NO: 172flanked by a putative left end with SEQ ID NO: 216 and a putative rightend with SEQ ID NO: 217; Pieris rapae (NCBI genomic reference sequenceNW_019093607.1, Genbank protein accession number XP_022123753.1) with anopen reading frame encoding a putative transposase with SEQ ID NO: 173flanked by a putative left end with SEQ ID NO: 218 and a putative rightend with SEQ ID NO: 219; Myzus persicae (NCBI genomic reference sequenceNW_019100532.1, protein accession number XP_022166603) with an openreading frame encoding a putative transposase with SEQ ID NO: 174flanked by a putative left end with SEQ ID NO: 220 and a putative rightend with SEQ ID NO: 221; Onthophagus taurus (NCBI genomic referencesequence NW_019280463, protein accession number XP_022900752) with anopen reading frame encoding a putative transposase with SEQ ID NO: 175flanked by a putative left end with SEQ ID NO: 222 and a putative rightend with SEQ ID NO: 223; Temnothorax curvispinosus (NCBI genomicreference sequence NW_020220783.1, protein accession numberXP_024881886) with an open reading frame encoding a putative transposasewith SEQ ID NO: 176 flanked by a putative left end with SEQ ID NO: 224and a putative right end with SEQ ID NO: 225; Agrlius planipenn (NCBIgenomic reference sequence NW_020442437.1, protein accession numberXP_025836109) with an open reading frame encoding a putative transposasewith SEQ ID NO: 177 flanked by a putative left end with SEQ ID NO: 226and a putative right end with SEQ ID NO: 227; Parasteatoda tepidariorum(NCBI genomic reference sequence NW_018371884.1, protein accessionnumber XP_015905033) with an open reading frame encoding a putativetransposase with SEQ ID NO: 178 flanked by a putative left end with SEQID NO: 228 and a putative right end with SEQ ID NO: 229; Pectinophoragossypiella (Genbank accession number GU270322.1, protein ID ADB45159.1,also described in Wang et al, 2010. Insect Mol. Biol. 19, 177-184.“piggyBac-like elements in the pink bollworm, Pectinophora gossypiella”)with an open reading frame encoding a putative transposase with SEQ IDNO: 179 flanked by a putative left end with SEQ ID NO: 230 and aputative right end with SEQ ID NO: 231; Ctenoplusia agnata (NCBIaccession number GU477713.1, protein accession number ADV17598.1, alsodescribed by Wu M, et al (2011) Genetica 139:149-54. “Cloning andcharacterization of piggyBac-like elements in lepidopteran insects”)with an open reading frame encoding a putative transposase with SEQ IDNO: 180 flanked by a putative left end with SEQ ID NO: 232 and aputative right end with SEQ ID NO: 233; Macrostomum lignano (NCBIgenomic reference sequence NIVC01003029.1, protein accession numberPAA53757) with an open reading frame encoding a putative transposasewith SEQ ID NO: 181 flanked by a putative left end with SEQ ID NO: 234and a putative right end with SEQ ID NO: 235; Orussus abietinus (NCBIaccession number XM_012421754, protein accession number XP_012277177)with an open reading frame encoding a putative transposase with SEQ IDNO: 182 flanked by a putative left end with SEQ ID NO: 236 and aputative right end with SEQ ID NO: 237; Eufriesea mexicana (NCBI genomicreference sequence NIVC01003029.1, protein accession numberXP_017759329) with an open reading frame encoding a putative transposasewith SEQ ID NO: 183 flanked by a putative left end with SEQ ID NO: 238and a putative right end with SEQ ID NO: 239; Spodoptera litura (NCBIgenomic reference sequence NC_036206.1, protein accession numberXP_022824855) with an open reading frame encoding a putative transposasewith SEQ ID NO: 184 flanked by a putative left end with SEQ ID NO: 240and a putative right end with SEQ ID NO: 241; Vanessa tameamea (NCBIgenomic reference sequence NW_020663261.1, protein accession numberXP_026490968) with an open reading frame encoding a putative transposasewith SEQ ID NO: 185 flanked by a putative left end with SEQ ID NO: 242and a putative right end with SEQ ID NO: 243; Blattella germanica (NCBIgenomic reference sequence PYGN01002011.1, protein accession numberPSN31819) with an open reading frame encoding a putative transposasewith SEQ ID NO: 186 flanked by a putative left end with SEQ ID NO: 244and a putative right end with SEQ ID NO: 245; Onthophagus taurus (NCBIgenomic reference sequence NW_019281532.1, protein accession numberXP_022910826) with an open reading frame encoding a putative transposasewith SEQ ID NO: 187 flanked by a putative left end with SEQ ID NO: 246and a putative right end with SEQ ID NO: 247; Onthophagus taurus (NCBIgenomic reference sequence NW_019281689.1, protein accession numberXP_022911139) with an open reading frame encoding a putative transposasewith SEQ ID NO: 188 flanked by a putative left end with SEQ ID NO: 248and a putative right end with SEQ ID NO: 249; Onthophagus taurus (NCBIgenomic reference sequence NW_019286114.1, protein accession numberXP_022913435) with an open reading frame encoding a putative transposasewith SEQ ID NO: 189 flanked by a putative left end with SEQ ID NO: 250and a putative right end with SEQ ID NO: 251; Megachile rotundata (NCBIgenomic reference sequence NW_003797295, protein accession numberXP_012145925) with an open reading frame encoding a putative transposasewith SEQ ID NO: 190 flanked by a putative left end with SEQ ID NO: 252and a putative right end with SEQ ID NO: 253; Xiphophorus maculatus(NCBI genomic reference sequence NC_036460.1, protein accession numberXP_023207869) with an open reading frame encoding a putative transposasewith SEQ ID NO: 191 flanked by a putative left end with SEQ ID NO: 254and a putative right end with SEQ ID NO: 255 and Amyelois transitella(NCBI accession number XM_013335311, protein accession numberXP_013190765.1) with an open reading frame encoding a putativetransposase with SEQ ID NO: 18 flanked by a putative left end with SEQID NO: 1 and a putative right end with SEQ ID NO: 3.

5.2.5.1 the Amyelois Transposase and its Corresponding Transposon

One active transposase and its corresponding transposon identified bytransposition activity in yeast was an Amyelois transposase, asdescribed in Section 6.1.2. An Amyelois transposase comprises apolypeptide sequence that is at least 80%, 90%, 93%, 95%, 96%, 97%, 98%,99% or 100% identical to the sequence given by SEQ ID NO: 18, and whichis capable of transposing the transposon from transposase reporterconstruct SEQ ID NO: 192, as described in Section 6.1.2. ExemplaryAmyelois transposases include sequences given as SEQ ID NOs: 18-170.

An Amyelois transposase may be provided as a part of a gene transfersystem as a protein, or as a polynucleotide encoding the Amyeloistransposase, wherein the polynucleotide is expressible in the targetcell. When provided as a polynucleotide, the Amyelois transposase may beprovided as DNA or mRNA. If provided as DNA, the open reading frameencoding the Amyelois transposase is preferably operably linked toheterologous regulatory elements including a promoter that is active inthe target cell such that the transposase is expressible in the targetcell, for example a promoter that is active in a eukaryotic cell or avertebrate cell or a mammalian cell. If provided as mRNA, the mRNA maybe prepared in vitro from a DNA molecule in which the open reading frameencoding the Amyelois transposase is preferably operably linked to aheterologous promoter active in the in vitro transcription system usedto prepare the mRNA, for example a T7 promoter.

An Amyelois transposon comprises a heterologous polynucleotide flankedby a left transposon end comprising a left ITR with sequence given bySEQ ID NO: 9 and a right transposon end comprising a right ITR withsequence given by SEQ ID NO: 10, and wherein the distal end of each ITRis immediately adjacent to a target sequence. Here and elsewhere wheninverted repeats are defined by a sequence including a nucleotidedefined by an ambiguity code, the identity of that nucleotide can beselected independently in the two repeats. A preferred target sequenceis 5′-TTAA-3′, although other useable target sequences may be used;preferably the target sequence on one side of the transposon is a directrepeat of the target sequence on the other side of the transposon. Theleft transposon end may further comprise additional sequences proximalto the ITR, for example a sequence at least 90% identical to, or 100%identical to SEQ ID NO: 7, or SEQ ID NO: 13. The right transposon endmay further comprise additional sequences proximal to the ITR, forexample a sequence at least 90% identical to, or 100% identical to SEQID NO: 8, or SEQ ID NO: 16. The structure of a representative Amyeloistransposon is shown in FIG. 1. An Amyelois transposon can be transposedby a transposase with a polypeptide sequence given by SEQ ID NO: 18, forexample as encoded by a polynucleotide with sequence given by SEQ ID NO:171 operably linked to a Gall promoter.

Transposon ends, including ITRs and target sequences may be added to theends of a heterologous polynucleotide sequence to create a syntheticAmyelois transposon which may be efficiently transposed into a targeteukaryotic genome by an Amyelois transposase. For example, SEQ ID NO: 1and SEQ ID NO: 2 each comprise a left 5′-TTAA-3′ target sequencefollowed by a left transposon ITR followed by additional end sequencesthat may be added to one side of a heterologous polynucleotide, with thetarget sequence distal relative to the heterologous polynucleotide, togenerate a synthetic Amyelois transposon. SEQ ID NO: 3, and SEQ ID NO: 4each comprise additional end sequences followed by a right transposonITR sequence followed by a right 5′-TTAA-3′ target sequence that may beadded to the other side of a heterologous polynucleotide, with thetarget sequence distal relative to the heterologous polynucleotide, togenerate a synthetic Amyelois transposon. The preceding transposon endsequences comprise 5′-TTAA-3′ as the target sequence, but SEQ ID NO: 5comprises a left transposon ITR followed by additional end sequencesthat may be added to one side of a heterologous polynucleotide, with theITR sequence distal relative to the heterologous polynucleotide, and SEQID NO: 6 comprises additional end sequences followed by a righttransposon ITR sequence that may be added to the other side of aheterologous polynucleotide, with the target sequence distal relative tothe heterologous polynucleotide, to generate a synthetic Amyeloistransposon which may then be flanked by alternative target sequences.

Amyelois transposases recognize synthetic Amyelois transposons. Theyexcise the transposon from a first DNA molecule, by cutting the DNA atthe target sequence at the left end of one transposon end and the targetsequence at the right end of the second transposon end, re-join the cutends of the first DNA molecule to leave a single copy of the targetsequence. The excised transposon sequence, including any heterologousDNA that is between the transposon ends, is integrated by thetransposase into a target sequence of a second DNA molecule, such as thegenome of a target cell. A cell whose genome comprises a syntheticAmyelois transposon is an embodiment of the invention.

5.2.5.2 the Amyelois Transposase is Active in Mammalian Cells

The looper moth piggyBac transposase has been shown to be active in avery wide variety of eukaryotic cells. In Section 6.1.2 we show that theAmyelois transposase can transpose its corresponding transposon into thegenome of the yeast Saccharomyces cerevisiae. In Section 6.1.3 we showthat the Amyelois transposase can transpose its corresponding transposoninto the genome of a mammalian CHO cell. These results provide evidencethat, like the other known active piggyBac-like transposases, theAmyelois transposase is also active in transposing its correspondingtransposon into the genomes of most eukaryotic cells. Although theAmyelois transposase is active in a wide range of eukaryotic cells, thenaturally occurring open reading frame encoding the Amyelois transposase(given by SEQ ID NO: 929) is unlikely to express well in a similarlywide range of cells, as optimal codon usage differs significantlybetween cell types. It is therefore advantageous to use a sequence otherthan a naturally occurring sequence to encode the transposase, in otherwords, to use codon-preferences of the cell type in which expression isto be performed. Likewise, the promoter and other regulatory sequencesare selected so as to be active in the cell type in which expression isto be performed. An advantageous polynucleotide for expression of anAmyelois transposase comprises at least 2, 5, 10, 20, 30, 40 or 50synonymous codon differences relative to SEQ ID NO: 929 at correspondingpositions between the polynucleotide and SEQ ID NO:929, optionallywherein codons in the polynucleotide at the corresponding positions areselected for mammalian cell expression. Exemplary polynucleotidesequences for Amyelois transposases with polypeptide sequences given bySEQ ID NOs: 18, 96 and 170 wherein codons have been selected forexpression in mammalian cells are given as SEQ ID NOs: 171, 930 and 931respectively. The polynucleotide may be DNA or mRNA.

5.2.6 Hyperactive Amyelois Transposases

Individual favorable mutations may be combined in a variety of differentways, for example by “DNA shuffling” or by methods described in U.S.Pat. No. 8,635,029 B2 or by methods described in U.S. Pat. No. 8,635,029B2 and Liao et al (2007, BMC Biotechnology 2007, 7:16doi:10.1186/1472-6750-7-16 “Engineering proteinase K using machinelearning and synthetic genes”). A transposase with modified activity,either for activity on a new target sequence, or increased activity onan existing target sequence may be obtained by using variations of theselection scheme described herein (for example Section 6.1.6) with anappropriate corresponding transposon.

An alignment of known active piggyBac-like transposases may be used toidentify amino acid changes likely to result in enhanced activity.Transposases are often deleterious to their hosts, so tend to accumulatemutations that inactivate them. However the mutations that accumulate indifferent transposases are different, as each occurs by random chance. Aconsensus sequence can be obtained from an alignment of sequences, andthis can be used to improve activity (Ivics et al, 1997. Cell 91:501-510. “Molecular reconstruction of Sleeping Beauty, a Tc1-liketransposon from fish, and its transposition in human cells.”). Wealigned known active piggyBac-like transposases using the CLUSTALalgorithm, and enumerated the amino acids found at each position. Thisdiversity is shown in Table 1 relative to an Amyelois transposase(relative to SEQ ID NO: 18), the amino acids shown in column C are foundin known active piggyBac-like transposases at the equivalent position inan alignment, and are thus likely to be acceptable changes in anAmyelois transposase. Column D shows amino acid changes found in knownactive piggyBac-like transposases other than the Amyelois transposase atpositions where there is good conservation within the rest of thetransposase set, but the amino acid in the Amyelois transposase sequenceis an outlier. Mutation of the position shown in column A to an aminoacid shown in column D is particularly likely to result in enhancedtransposase activity.

We selected 59 amino acid substitutions to make in Amyelois transposaseSEQ ID NO: 18 from column D in Table 1. The substitutions were P65E,D79N, R95S, V100I, V106P, L115D, E116P, H121Q, V131E, K139E, T159N,V166F, G174A, G179N, W187F, P198R, L203R, I209L, N211R, A225R, P232A,E238D, V261L, L273M, L273I, D304R, D304K, Y310G, I323L, Q329G, Q329R,M336C, Y343I, T345L, K362R, T366R, C367E, T380S, L408M, E413S, S416E,I426M, K432Q, S435G, K442Q, N452K, L458M, N466D, T467M, A472S, V475I,N483K, I491M, A529P, K540R, D551R, S560K, T562K and S563K. Genesencoding Amyelois transposase variants comprising combinations of thesesubstitutions were synthesized and tested for transposase activity asdescribed in Section 6.1.6. We identified more than 70 Amyeloistransposase variants with increased excision or transposition activitycompared with the naturally occurring sequence SEQ ID NO: 18. Exemplarysequences of hyperactive Amyelois transposase variants are provided asSEQ ID NOs: 96-170. Particularly active Amyelois transposase variantsare provided as SEQ ID NOs: 114-170.

Amyelois transposases can thus be created that are not naturallyoccurring sequences, but that are at least 99%, 98%, 97%, 96%, 95%, 90%or at least 80% identical to SEQ ID NO 18. Such variants can retainpartial activity of the transposase of SEQ ID NO: 18 (as determined byeither or both of transposition and/or excision activity), can befunctionally equivalent of the transposase of SEQ ID NO: 18 in either orboth of transposition and excision, or can have enhanced activityrelative to the transposase of SEQ ID NO: 18 in transposition, excisionactivity or both. Such variants can include mutations shown herein toincrease transposition and/or excision, mutations shown herein to beneutral as to transposition and/or excision, and mutations detrimentalto transposition and/or integration. Preferred variants includemutations shown to be neutral or to enhance transposition/and orexcision. Some such variants lack mutations shown to be detrimental totransposition and/or excision. Some such variants include only mutationsshown to enhance transposition, only mutations shown to enhanceexcision, or mutations shown to enhance both transposition and excision.

Enhanced activity means activity (e.g., transposition or excisionactivity) that is greater beyond experimental error than that of areference transposase from which a variant was derived. The activity canbe greater by a factor of e.g., 1.5, 2, 5, 10, 15, 20, 50 or 100 fold ofthe reference transposase. The enhanced activity can lie within a rangeof for example 2-100 fold, 2-50 fold, 5-50 fold or 2-10 fold of thereference transposase. Here and elsewhere activities can be measured asdemonstrated in the examples.

Functional equivalence means a variant transposase can mediatetransposition and/or excision of the same transposon with a comparableefficiency (within experimental error) to a reference transposase.Seventeen representative sequences of variant Amyelois transposases withtransposition frequencies comparable to naturally occurring Amyeloistransposase SEQ ID NO: 18 are SEQ ID NOs: 19-35.

Furthermore, variant sequences of SEQ ID NO:18 can be created bycombining two, three, four, or five or more substitutions selected fromTable 1 column D. Combining beneficial substitutions, for example thoseshown in column D of Table 1 can result in hyperactive variants of SEQID NO: 18. We identified more than 70 Amyelois transposases (comprisingsequences provided as SEQ ID NOs: 96-170) with transposition or excisionfrequencies more than about 2-fold greater than that of naturallyoccurring Amyelois transposase SEQ ID NO: 18. These preferredhyperactive Amyelois transposases comprised one or more of the followingsubstitutions (relative to SEQ ID NO: 18): D79N, R95S, L115D, E116P,H121Q, K139E, V166F, G179N, W187F, P198R, L203R, N211R, E238D, L273M,L273I, D304R, D304K, Q329G, T345L, K362R, T366R, L408M, S416E, S435G,L458M, V475I, N483K, I491M, A529P, K540R, S560K and S563K. Somehyperactive Amyelois transposases may further comprise a heterologousnuclear localization sequence.

We used machine learning methods as described in Liao et al (2007, BMCBiotechnology 2007, 7:16 doi:10.1186/1472-6750-7-16 “Engineeringproteinase K using machine learning and synthetic genes”) to determinethe effect of various amino acid substitutions on the excision andtransposition activities of an Amyelois transposase. Each substitutionwas empirically tested in a minimum of 5 different sequence contexts(i.e. in the presence of different other amino acid substitutions). Asdescribed in Liao et. al., the mean value for the regression weight fora substitution is a measure of the average effect of that substitutionwithin multiple different Amyelois transposases. A substitution with apositive mean regression weight is one that on average has a positiveeffect on the transposition activity of Amyelois transposases. Additionof a substitution with a positive mean regression weight to an activeAmyelois transposase that does not already comprise such a substitutionis thus expected to improve the transposition activity of that Amyeloistransposase. Table 8 identifies 32 substitutions within an Amyeloistransposase with positive mean regression weights for transposition:D79N, R95S, L115D, E116P, H121Q, K139E, V166F, G179N, W187F, P198R,L203R, N211R, E238D, L273M, L273I, D304R, D304K, Q329G, T345L, K362R,T366R, L408M, S416E, S435G, L458M, V475I, N483K, I491M, A529P, K540R,S560K and S563K.

SEQ ID NOs: 18-35 or 96-170 and comprise a substitution at a positionselected from amino acid 79, 95, 115, 116, 121, 139, 166, 179, 187, 198,203, 211, 238, 273, 304, 329, 345, 362, 366, 408, 416, 435, 458, 475,483, 491, 529, 540, 560 and 563, relative to SEQ ID NO: 18. Preferablythe substitution is one shown in Table 1 columns C or D. Preferably thehyperactive Amyelois transposase comprises an amino acid substitution,relative to the sequence of SEQ ID NO: 18, selected from D79N, R95S,L115D, E116P, H121Q, K139E, V166F, G179N, W187F, P198R, L203R, N211R,E238D, L273M, L273I, D304R, D304K, Q329G, T345L, K362R, T366R, L408M,S416E, S435G, L458M, V475I, N483K, I491M, A529P, K540R, S560K and S563K,or any combination of substitutions thereof including at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or all of these mutations. The hyperactive Amyeloistransposase may also comprise substitutions at many other positions thatare not associated with enhanced transposition, for example conservativesubstitutions that have a neutral effect on transposition.

Preferred hyperactive Amyelois transposases comprise an amino acidsequence, other than a naturally occurring protein (e.g., not atransposase whose amino acid sequence comprises SEQ ID NO: 18), that isat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of any of SEQ ID NOs: 18-35 or 96-170 and comprise asubstitution at a position selected from amino acid 79, 95, 115, 116,121, 139, 166, 179, 187, 198, 203, 211, 238, 273, 304, 329, 345, 362,366, 408, 416, 435, 458, 475, 483, 491, 529, 540, 560 and 563, relativeto SEQ ID NO: 18. Preferably the substitution is one shown in Table 1columns C or D. Preferably the hyperactive Amyelois transposasecomprises an amino acid substitution, relative to the sequence of SEQ IDNO: 18, selected from D79N, R95S, L115D, E116P, H121Q, K139E, V166F,G179N, W187F, P198R, L203R, N211R, E238D, L273M, L273I, D304R, D304K,Q329G, T345L, K362R, T366R, L408M, S416E, S435G, L458M, V475I, N483K,I491M, A529P, K540R, S560K and S563K, or any combination ofsubstitutions thereof including at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or all of these mutations.

Methods of creating transgenic cells using naturally occurring orhyperactive Amyelois transposases are an aspect of the invention. Amethod of creating a transgenic cell comprises (i) introducing into aeukaryotic cell a naturally occurring or hyperactive Amyeloistransposase (as a protein or as a polynucleotide encoding thetransposase) and a corresponding Amyelois transposon. Creating thetransgenic cell may further comprise (ii) identifying a cell in which anAmyelois transposon is incorporated into the genome of the eukaryoticcell. Identifying the cell in which an Amyelois transposon isincorporated into the genome of the eukaryotic cell may compriseselecting the eukaryotic cell for a selectable marker encoded on theAmyelois transposon. The selectable marker may be any selectablepolypeptide, including any described herein.

Activity of transposases may also be increased by fusion of nuclearlocalization signal (NLS) at the N-terminus, C-terminus, both at the N-and C-termini or internal regions of the transposase protein, as long astransposase activity is retained. A nuclear localization signal orsequence (NLS) is an amino acid sequence that ‘tags’ or facilitatesinteraction of a protein, either directly or indirectly with nucleartransport proteins for import into the cell nucleus. Nuclearlocalization signals (NLS) used can include consensus NLS sequences,viral NLS sequences, cellular NLS sequences, and combinations thereof.

Transposases may also be fused to other protein functional domains. Suchprotein functional domains can include DNA binding domains, flexiblehinge regions that can facilitate one or more domain fusions, andcombinations thereof. Fusions can be made either to the N-terminus,C-terminus, or internal regions of the transposase protein so long astransposase activity is retained. Fusions to DNA binding domains can beused to direct the Amyelois transposase to a specific genomic locus orloci. DNA binding domains may include a helix-turn-helix domain, azinc-finger domain, a leucine zipper domain, a TALE (transcriptionactivator-like effector) domain, a CRISPR-Cas protein or ahelix-loop-helix domain. Specific DNA binding domains used can include aGal4 DNA binding domain, a LexA DNA binding domain, or a Zif268 DNAbinding domain. Flexible hinge regions used can include glycine/serinelinkers and variants thereof.

5.3 Kits

The present invention also features kits comprising an Amyeloistransposase as a protein or encoded by a nucleic acid, and/or anAmyelois transposon; or a gene transfer system as described hereincomprising an Amyelois transposase as a protein or encoded by a nucleicacid as described herein, in combination with an Amyelois transposon;optionally together with a pharmaceutically acceptable carrier, adjuvantor vehicle, and optionally with instructions for use. Any of thecomponents of the inventive kit may be administered and/or transfectedinto cells in a subsequent order or in parallel, e.g. an Amyeloistransposase protein or its encoding nucleic acid may be administeredand/or transfected into a cell as defined above prior to, simultaneouslywith or subsequent to administration and/or transfection of an Amyeloistransposon. Alternatively, an Amyelois transposon may be transfectedinto a cell as defined above prior to, simultaneously with or subsequentto transfection of an Amyelois transposase protein or its encodingnucleic acid. If transfected in parallel, preferably both components areprovided in a separated formulation and/or mixed with each otherdirectly prior to administration to avoid transposition prior totransfection. Additionally, administration and/or transfection of atleast one component of the kit may occur in a time staggered mode, e.g.by administering multiple doses of this component.

6. EXAMPLES

The following examples illustrate the methods, compositions and kitsdisclosed herein and should not be construed as limiting in any way.Various equivalents will be apparent from the following examples; suchequivalents are also contemplated to be part of the invention disclosedherein.

6.1 a New Transposase

6.1.1 Measuring Transposase Activity

As described in Section 5.2.5, transposition frequencies for activetransposases may be measured using a system in which a transposoninterrupts a selectable marker. Transposase reporter polynucleotideswere constructed in which the open reading frame of the yeastSaccharomyces cerevisiae URA3 open reading frame was interrupted by ayeast TRP 1 open reading frame operably linked to a promoter andterminator such that it was expressible in the yeast Saccharomycescerevisiae. The TRP1 gene was flanked by putative transposon ends with5′-TTAA-3′ target sites, such that excision of the putative transposonwould leave a single copy of the 5′-TTAA-3′ target site and exactlyreconstitute the URA3 open reading frame. A yeast transposase reporterstrain was constructed by integrating the transposase reporterpolynucleotide into the URA3 gene of a haploid yeast strain auxotrophicfor LEU2 and TRP1, such that the strain became LEU2−, URA3− and TRP1+.

Transposases were tested for their ability to transposase the TRP1gene-containing transposons from within the URA3 open reading frame.Each open reading frame encoding a putative transposase was cloned intoa Saccharomyces cerevisiae expression vector comprising a 2 micronorigin of replication and a LEU2 gene expressible in Saccharomyces. Eachtransposase open reading frame was operably linked to a Gall promoter.Each cloned transposase open reading frame was transformed into a yeasttransposase reporter strain and plated on minimal media lacking leucine.After 2 days, all LEU+ colonies were harvested by scraping the plates.The Gal promoter was induced by growing in galactose for 4 hours, andcells were then plated onto 3 different plates: plates lacking onlyleucine, plates lacking leucine and uracil, and plates lacking leucine,uracil and tryptophan. These plates were incubated for 2-4 days, and thecolonies on each plate were counted, measuring the number of live cells,the number of transposon excision events and the number of transposonexcision and re-integration (i.e. transposition events) respectively.

6.1.2 Identification of an Active Amyelois PiggyBac-Like Transposase

As described in Section 5.2.5, twenty-one putative piggyBac-liketransposases were identified from Genbank as being at least 20%identical to the piggyBac transposase from Trichoplusia ni. Theseputative transposases appeared to comprise the DDDE motif characteristicof active piggyBac-like transposases. The flanking DNA sequences wereanalyzed for the presence of inverted repeat sequences immediatelyadjacent to the 5′-TTAA-3′ target sequence characteristic of piggyBactransposition. Putative left and right transposon end sequencescomprising the sequence between the 5′-TTAA-3′ target sequence and theopen reading frame encoding the putative transposase were taken fromthese flanking sequences. These transposon ends were incorporated intotransposase reporter constructs configured as described in Section 6.1.1and integrated into the genome of Saccharomyces cerevisiae therebygenerating transposase reporter strains. The corresponding transposasesequence for each reporter strain was back-translated, synthesized,cloned into a Saccharomyces cerevisiae expression vector and transformedinto the reporter strain. Transposase activities were measured asdescribed in Section 6.1.1.

The following twenty combinations showed no excision or transposition:reporter construct SEQ ID NO: 196 (comprising putative left transposonend SEQ ID NO: 216, and putative right transposon end SEQ ID NO: 217)with transposase SEQ ID NO: 172, reporter construct SEQ ID NO: 197(comprising putative left transposon end SEQ ID NO: 218, and putativeright transposon end SEQ ID NO: 219) with transposase SEQ ID NO: 173,reporter construct SEQ ID NO: 198 (comprising putative left transposonend SEQ ID NO: 220, and putative right transposon end SEQ ID NO: 221)with transposase SEQ ID NO: 174, reporter construct SEQ ID NO: 199(comprising putative left transposon end SEQ ID NO: 222, and putativeright transposon end SEQ ID NO: 223) with transposase SEQ ID NO: 175,reporter construct SEQ ID NO: 200 (comprising putative left transposonend SEQ ID NO: 224, and putative right transposon end SEQ ID NO: 225)with transposase SEQ ID NO: 176, reporter construct SEQ ID NO: 201(comprising putative left transposon end SEQ ID NO: 226, and putativeright transposon end SEQ ID NO: 227) with transposase SEQ ID NO: 177,reporter construct SEQ ID NO: 202 (comprising putative left transposonend SEQ ID NO: 228, and putative right transposon end SEQ ID NO: 229)with transposase SEQ ID NO: 178, reporter construct SEQ ID NO: 203(comprising putative left transposon end SEQ ID NO: 230, and putativeright transposon end SEQ ID NO: 231) with transposase SEQ ID NO: 179,reporter construct SEQ ID NO: 204 (comprising putative left transposonend SEQ ID NO: 232, and putative right transposon end SEQ ID NO: 233)with transposase SEQ ID NO: 180, reporter construct SEQ ID NO: 205(comprising putative left transposon end SEQ ID NO: 234, and putativeright transposon end SEQ ID NO: 235) with transposase SEQ ID NO: 181,reporter construct SEQ ID NO: 206 (comprising putative left transposonend SEQ ID NO: 236 and putative right transposon end SEQ ID NO: 237)with transposase SEQ ID NO: 182, reporter construct SEQ ID NO: 207(comprising putative left transposon end SEQ ID NO: 238, and putativeright transposon end SEQ ID NO: 239) with transposase SEQ ID NO: 183,reporter construct SEQ ID NO: 208 (comprising putative left transposonend SEQ ID NO: 240, and putative right transposon end SEQ ID NO: 241)with transposase SEQ ID NO: 184, reporter construct SEQ ID NO: 209(comprising putative left transposon end SEQ ID NO: 242, and putativeright transposon end SEQ ID NO: 243) with transposase SEQ ID NO: 185,reporter construct SEQ ID NO: 210 (comprising putative left transposonend SEQ ID NO: 244, and putative right transposon end SEQ ID NO: 245)with transposase SEQ ID NO: 186, reporter construct SEQ ID NO: 211(comprising putative left transposon end SEQ ID NO: 246, and putativeright transposon end SEQ ID NO: 247) with transposase SEQ ID NO: 187,reporter construct SEQ ID NO: 212 (comprising putative left transposonend SEQ ID NO: 248, and putative right transposon end SEQ ID NO: 249)with transposase SEQ ID NO: 188, reporter construct SEQ ID NO: 213(comprising putative left transposon end SEQ ID NO: 250, and putativeright transposon end SEQ ID NO: 251) with transposase SEQ ID NO: 189,reporter construct SEQ ID NO: 214 (comprising putative left transposonend SEQ ID NO: 252, and putative right transposon end SEQ ID NO: 253)with transposase SEQ ID NO: 190, reporter construct SEQ ID NO: 215(comprising putative left transposon end SEQ ID NO: 254 and putativeright transposon end SEQ ID NO: 255) with transposase SEQ ID NO: 191.This is consistent with reports in the literature that whilecomputational recognition of sequences that are homologous to thepiggyBac transposase from Trichoplusia ni is straightforward, most ofthese sequences are transpositionally inactive, even when they appear tohave intact terminal repeats and the transposases appear to comprise theDDDE motif found in active piggyBac-like transposases. It is thereforenecessary to measure excision and transposition activity, in order toidentify novel active piggyBac-like transposases and transposons.

One transposase that showed good activity in excising its correspondingtransposon from the reporter construct (shown by the appearance ofURA+colonies) and transposing the TRP gene in the transposon intoanother genomic location in the Saccharomyces cerevisiae reporter strainwas transposase SEQ ID NO: 18. Transposase SEQ ID NO: 18 was able totranspose the transposon from reporter construct SEQ ID NO: 192. This isshown in Table 2: the number of excision events, measured by theappearance of URA+ colonies, is shown in column G; the number of fulltransposition events, measured by the appearance of URA+ TRP+ colonies,is shown in column H.

6.1.3 the Amyelois Transposase is Active in Mammalian Cells

PiggyBac-like transposases can transpose their corresponding transposonsinto the genomes of eukaryotic cells including yeast cells such asPichia pastoris and Saccharomyces cerevisiae, and mammalian cells suchas human embryonic kidney (HEK) and Chinese hamster ovary (CHO) cells.To determine the activity of piggyBac-like transposases in mammaliancells, we constructed gene transfer polynucleotides comprisingtransposon ends, and further comprising a selectable marker encodingglutamine synthetase with a polypeptide sequence given by SEQ ID NO:277, encoded by DNA sequence given by SEQ ID NO: 282 and operably linkedto regulatory elements that give weak glutamine synthetase expression,the sequence of the glutamine synthetase and its associated regulatoryelements given by SEQ ID NO: 320. The gene transfer polynucleotidesfurther comprised open reading frames encoding the heavy and lightchains of an antibody, each operably linked to a promoter andpolyadenylation signal sequence. The gene transfer polynucleotide (withSEQ ID NO: 256) comprised a left transposon end comprising a 5′-TTAA-3′target integration sequence immediately followed by an Amyelois lefttransposon end with ITR sequence given by SEQ ID NO: 11, which is anembodiment of SEQ ID NO: 9. The gene transfer polynucleotide furthercomprised an Amyelois right transposon end with ITR sequence given bySEQ ID NO: 12 (which is an embodiment of SEQ ID NO: 10) immediatelyfollowed by a 5′-TTAA-3′ target integration sequence. The two Amyeloistransposon ends were placed on either side of the heterologouspolynucleotide comprising the glutamine synthetase selectable marker andthe open reading frames encoding the heavy and light chains of theantibody. The left transposon end further comprised a sequence given bySEQ ID NO: 7 immediately adjacent to the left ITR and proximal to theheterologous polynucleotide. The right transposon end further compriseda sequence given by SEQ ID NO: 8 immediately adjacent to the right ITRand proximal to the heterologous polynucleotide.

Gene transfer polynucleotides were transfected into CHO cells whichlacked a functional glutamine synthetase gene. Cells were transfected byelectroporation with 25 μg of gene transfer polynucleotide DNA, eitherwith or without a co-transfection with 3 μg of DNA comprising a geneencoding a transposase operably linked to a human CMV promoter and apolyadenylation signal sequence. The cells were incubated in mediacontaining 4 mM glutamine for 48 hours following electroporation, andsubsequently diluted to 300,000 cells per ml in media lacking glutamine.Cells were exchanged into fresh glutamine-free media every 5 days. Theviability of the cells from each transfection were measured at varioustimes following transfection using a Beckman-Coulter Vi-Cell. The totalnumber of viable cells were also measured with the same instrument. Theresults are shown in Table 3.

As shown in Table 3, the viability of cells transfected with the genetransfer polynucleotide, but no transposase fell to about 27% by 12 dayspost-transfection (column B). The total number of live cells fell tofewer than 50,000 per ml within 7 days (column C). At or below thisdensity of live cells, viability measurements become inaccurate. Theculture never recovered. In contrast when gene transfer polynucleotidewith SEQ ID NO: 256 was co-transfected with Amyelois transposase SEQ IDNO: 18, cells recovered to greater than 90% viability within 17 days(Table 3 column D), by which time the density of live cells exceeded 2million per ml (Table 3 column E). This shows that a gene transferpolynucleotide comprising a left and right Amyelois transposon end canbe efficiently transposed into the genome of a mammalian target cell bya corresponding Amyelois transposase.

The recovered pools of CHO cells comprising piggyBac-like transposonsintegrated into their genomes were grown in a 14-day fed-batch usingSigma Advanced Fed Batch media. Antibody titers were measured in culturesupernatant using an Octet. Table 4 shows the titers measured at 7, 10,12 and 14 days of the fed batch culture. The titer of antibody fromcells comprising gene transfer polynucleotide with SEQ ID NO: 256, thathad been integrated by co-transfection with the Amyelois transposase SEQID NO: 18 reached approximately 2 g/L after 14 days. This shows that theAmyelois transposon and its corresponding transposase, as described inSection 5.2.5, is a novel, piggyBac-like transposon/transposase systemthat is active in mammalian cells and useful for developing proteinexpressing cell lines and engineering the genomes of mammalian cells.

6.1.4 Messenger RNA Encoding the Amyelois Transposase is Active inMammalian Cells

We further tested gene transfer polynucleotide with SEQ ID NO: 256,whose configuration is described in Section 6.1.3, to determine whetherthe synthetic Amyelois transposon could be integrated into the genome ofa mammalian cell if the corresponding transposase was provided in theform of mRNA. Gene transfer polynucleotide 354498 with SEQ ID NO: 256comprised a selectable marker encoding glutamine synthetase with apolypeptide sequence given by SEQ ID NO: 277, encoded by DNA sequencegiven by SEQ ID NO: 282 and operably linked to regulatory elements thatgive weak glutamine synthetase expression, the sequence of the glutaminesynthetase and its associated regulatory elements given by SEQ ID NO:320. Gene transfer polynucleotide SEQ ID NO: 256 further comprised openreading frames encoding the heavy and light chains of an antibody, eachoperably linked to a promoter and polyadenylation signal sequence. Genetransfer polynucleotide SEQ ID NO: 256 further comprised an Amyeloisleft transposon end with sequence given by SEQ ID NO: 1 and an Amyeloisright transposon end with sequence given by SEQ ID NO: 3.

mRNA encoding Amyelois transposase was prepared by in vitrotranscription using T7 RNA polymerase. The mRNA comprised a 5′ sequenceSEQ ID NO: 257 preceding the open reading frame, an open reading frameencoding an Amyelois transposase (amino acid sequence SEQ ID NO: 18,nucleotide sequence SEQ ID NO: 171), and a 3′ sequence SEQ ID NO: 258following the stop codon at the end of the open reading frame. The mRNAhad an anti-reverse cap analog (3′-O-Me-m⁷G(5′)ppp(5′)G. DNA moleculescomprising a sequence encoding a transposase operably linked to aheterologous promoter that is active in vitro are useful for thepreparation of transposase mRNA. Isolated mRNA molecules comprising asequence encoding a transposase are useful for transposition of acorresponding transposon into a target genome.

Gene transfer polynucleotide SEQ ID NO: 256 was transfected into CHOcells which lacked a functional glutamine synthetase gene. Cells weretransfected by electroporation: 25 μg of gene transfer polynucleotideDNA was co-transfected with 3 μg of mRNA comprising an open readingframe encoding a corresponding transposase (amino acid sequence SEQ IDNO: 18, nucleotide sequence SEQ ID NO: 171). The cells were incubated inmedia containing 4 mM glutamine for 48 hours following electroporation,and subsequently diluted to 300,000 cells per ml in media lackingglutamine. Cells were exchanged into fresh glutamine-free media every 5days. The viability of the cells from each transfection were measured atvarious times following transfection using a Beckman-Coulter Vi-Cell.The total number of viable cells were also measured with the sameinstrument. The results are shown in Table 5.

When gene transfer polynucleotide with SEQ ID NO: 256 was co-transfectedwith mRNA encoding Amyelois transposase SEQ ID NO: 18, viability fell toaround 26% by 9 days post-transfection (Table 5 column B), by which timethe density of live cells was around 50,000 per ml (Table 5 column C).Cell viability and the density of live cells then increased until by 26days post-transfection viability was above 92% and there were over 1million live cells per ml. This shows that a gene transferpolynucleotide comprising a left and right Amyelois transposon end canbe efficiently transposed into the genome of a mammalian target cellwhen co-transfected with mRNA encoding a corresponding Amyeloistransposase.

6.1.5 Amyelois Transposon End Sequences Active in Mammalian Cells

When we originally tested the Amyelois transposon, we used the entiresequence between the 5′-TTAA-3′ target sequences and the transposaseopen reading frame as transposon ends. We have found that for otherpiggyBac-like sequences this full sequence is generally not required fortransposition activity. We therefore constructed synthetic Amyeloistransposons with truncated ends to determine whether these weretransposable by an Amyelois transposase. A heterologous polynucleotidewith SEQ ID NO: 370 encoded glutamine synthetase with a polypeptidesequence given by SEQ ID NO: 278, operably linked to regulatory elementsthat give weak glutamine synthetase expression as a selectable marker.On one side of the heterologous polynucleotide was a left Amyeloistransposon end comprising a 5′-TTAA-3′ integration target sequenceimmediately followed by a transposon ITR sequence with SEQ ID NO: 11,which is an embodiment of SEQ ID NO: 9. On the other side of theheterologous polynucleotide was a right Amyelois transposon endcomprising a transposon ITR sequence with SEQ ID NO: 12 (which is anembodiment of SEQ ID NO: 10) immediately followed by a 5′-TTAA-3′integration target sequence. The transposon further comprised anadditional sequence selected from SEQ ID NOs: 7 or 13 immediatelyadjacent to (following) the left transposon ITR sequence. The transposonfurther comprised an additional sequence selected from SEQ ID NOs: 8 or16 immediately adjacent to (preceding) the right transposon ITRsequence. Transposons were transfected into CHO cells which lacked afunctional glutamine synthetase gene. Cells were transfected byelectroporation: 25 μg of gene transfer polynucleotide DNA weretransfected, optionally the cells were co-transfected with 3 μg of mRNAcomprising an open reading frame encoding a corresponding transposase(amino acid sequence SEQ ID NO: 18, nucleotide sequence SEQ ID NO: 171).The cells were incubated in media containing 4 mM glutamine for 48 hoursfollowing electroporation, and subsequently diluted to 300,000 cells perml in media lacking glutamine. Cells were exchanged into freshglutamine-free media every 5 days. The viability of the cells from eachtransfection were measured at various times following transfection usinga Beckman-Coulter Vi-Cell. The total number of viable cells were alsomeasured with the same instrument. The results are shown in Table 6.

Table 6 columns B and C show the reduction in cell viability and viablecell density when cells were transfected with a transposon comprisingfull length transposon ends in the absence of transposase. Cellviability and viable cell density can both be seen to fall throughoutthe experiment. In contrast when any the same transposon wasco-transfected with mRNA encoding an Amyelois transposase, the cellviability and viable cell density fell initially, but had begun torecover by day 18 and was fully recovered between day 25 and 29 (Table 6columns C and D). A comparable result was obtained when the lefttransposon end was truncated from the sequence given by SEQ ID NO: 7, tothe sequence given by SEQ ID NO: 13 (compare Table 6 columns E and Fwith columns G and H respectively). A comparable result was alsoobtained when the right transposon end was truncated from the sequencegiven by SEQ ID NO: 8, to the sequence given by SEQ ID NO: 16 (compareTable 6 columns I and J with columns K and L respectively). This showsthat in addition to a 5′-TTAA-3′ integration target sequence immediatelyadjacent to a transposon ITR sequence with SEQ ID NO: 9, an Amyeloissynthetic transposon left transposon end may further comprise anadditional sequence selected from SEQ ID NOs: 7 and 13 immediatelyadjacent to the left transposon ITR sequence; and an Amyelois synthetictransposon right transposon end may comprise an additional sequenceselected from SEQ ID NOs: 8 and 16 immediately adjacent to the righttransposon ITR sequence.

6.1.6 Engineering Hyperactive Amyelois Transposases

To identify Amyelois transposase mutations that led to either increasedtransposition activity, or increased excision activity, relative to thenaturally occurring Amyelois transposase sequence given by SEQ ID NO:18, we analyzed a CLUSTAL alignment of active piggyBac-liketransposases. Table 1 column C shows the amino acids found in activepiggyBac-like transposases relative to each position in the Amyeloistransposase (position shown in Table 1 column A). The amino acid presentin Amyelois transposase given by SEQ ID NO: 18 is shown in column B ofTable 1. Because transposases are often deleterious to their hosts, theytend to accumulate mutations that inactivate them. The mutations thataccumulate in different transposases are different, as each occurs byrandom chance. A consensus sequence can therefore be used to approximatean ancestral sequence that pre-dates the accumulation of deleteriousmutations. It is difficult to accurately calculate an ancestral sequencefrom a small number of extant sequences, so we chose to focus onpositions where active transposases were more highly conserved, andwhere the consensus amino acid(s) differed from the one in the Amyeloistransposase. We considered that mutating these amino acids to theconsensus amino acids found in other active transposases would be likelyto increase the activity of the Amyelois transposase. These candidatebeneficial amino acid substitutions are shown in Table 1 column D.

6.1.6.1 First Set of Amyelois Transposase Variants

A set of 95 genes encoding variant Amyelois transposases comprised oneor more substitutions selected from P65E, D79N, R95S, V100I, V106P,L115D, E116P, H121Q, V131E, K139E, T159N, V166F, G174A, G179N, W187F,P198R, L203R, I209L, N211R, A225R, P232A, E238D, V261L, L273M, L273I,D304R, D304K, Y310G, I323L, Q329G, Q329R, M336C, Y343I, T345L, K362R,T366R, C367E, T380S, L408M, E413S, S416E, I426M, K432Q, S435G, K442Q,N452K, L458M, N466D, T467M, A472S, V475I, N483K, I491M, A529P, K540R,D551R, S560K, T562K and S563K. Each substitution was represented atleast 5 times within the set of 96 variants, and the number of differentpairwise combinations of substitutions was maximized so that eachsubstitution was tested in as many different sequence contexts aspossible. Each variant gene was cloned into a vector comprising aleucine selectable marker; each gene encoding a transposase variant wasoperably linked to the Saccharomyces cerevisiae Gal-1 promoter. Each ofthese variants was then individually transformed into a Saccharomycescerevisiae strain comprising a chromosomally integrated copy of SEQ IDNO: 192, as described above. After 48 hours cells were scraped from theplate into minimal media lacking leucine and with galactose as thecarbon source. The A600 for each culture was adjusted to 2. Cultureswere grown for 4 hours in galactose to induce expression of thetransposases, then a 100×-diluted aliquot was plated on media lackingleucine, uracil and tryptophan (to count transposition), a 100×-dilutedaliquot was plated on media lacking leucine and uracil (to countexcision) and a 25,000×-diluted aliquot was plated on media lackingleucine (to count total live cells). Two days later, colonies werecounted to determine transposition (=number of cells on -leu-ura-trpmedia divided by (250×number of cells on -leu media)) and excision(=number of cells on -leu-ura media divided by (250×number of cells on-leu media)) frequencies. The results are shown in Table 7. Weidentified 18 Amyelois transposase variants (with sequences given by SEQID NO: 96-113) with excision or transposition activities that werebetween 1.2 and 5-fold higher than the activities measured for thenaturally occurring Amyelois transposase. We also identified 17 Amyeloistransposase variants (with sequences given by SEQ ID NO: 19-35) withexcision or transposition activities that were comparable to thenaturally occurring Amyelois transposase. We also identified 45 Amyeloistransposase variants (with sequences given by SEQ ID NO: 51-95) withexcision or transposition activities that were less than the naturallyoccurring Amyelois transposase. Only 15 of the 95 variants (withsequences given by SEQ ID NO: 36-50) possessed activities that were solow that they were essentially inactive.

The effects of sequence changes on transposition frequencies weremodelled using two different methods as described in Liao et al (2007,BMC Biotechnology 2007, 7:16 doi:10.1186/1472-6750-7-16 “Engineeringproteinase K using machine learning and synthetic genes”) and U.S. Pat.No. 8,635,029. Mean values and standard deviations for the regressionweights were calculated for each substitution, these are shown in Table8. The effect of an individual substitution upon transposase activitymay vary depending on the context (ie the other substitutions present).A positive mean regression weight indicates that on average, consideringall of the different sequence contexts in which it has been tested, thesubstitution has a positive influence on the measured property.Incorporation of substitutions with positive mean regression weightsinto a sequence generally results in variants with improved activity(Liao et. al., ibid). A further measure of this context-dependentvariability is the standard deviation of the regression weight. If themean regression weight plus the standard deviation of the regressionweight for a substitution is zero or greater, then there are contextswithin which the substitution has a positive effect. If the meanregression weight for a substitution is zero or greater, then theaverage effect of the substitution in all measured contexts is positive(Liao et al, ibid). If the mean regression weight minus the standarddeviation of the regression weight for a substitution is zero orgreater, then the substitution has a positive effect in the majority ofsequence contexts (Liao et al, ibid). Thirty-three of the fifty ninesubstitutions we selected by looking for changes toward the consensus inother active piggyBac-like transposases had a mean regression weight ofzero or greater as determined by one of the methods used: D79N, R95S,L115D, E116P, H121Q, K139E, V166F, G179N, W187F, P198R, L203R, N211R,E238D, L273M, L273I, D304R, D304K, Q329G, T345L, K362R, T366R, L408M,S416E, S435G, L458M, V475I, N483K, I491M, A529P, K540R, S560K, S563K. Inaddition to identifying specific substitutions with a beneficial effect,this also provides an indication of positions at which analogoussubstitutions may be beneficial. For example, replacement of the acidicaspartate at position 304 with either the basic residue lysine or thebasic residue arginine has a positive effect. This provides evidencethat multiple different analogous substitutions at a position can bebeneficial. Analogous substitutions are those in which properties of theamino acids are conserved. For example: glycine and alanine are in the“small” amino acid group; valine, leucine, isoleucine and methionine arein the “hydrophobic” amino acid group; phenylalanine, tyrosine andtryptophan are in the “aromatic” amino acid group; aspartate andglutamate are in the “acidic” amino acid group; asparagine and glutamineare in the “amide” amino acid group; histidine, lysine and arginine arein the “basic” amino acid group; cysteine, serine and threonine are inthe “nucleophilic” amino acid group. If a substitution at an amino acidposition within the Amyelois transposase is beneficial for excision ortransposition activity, other substitutions at the same position drawnfrom the same amino acid group are likely to be beneficial. For example,since replacing the basic residue lysine at position 139 with the acidicresidue glutamate (K139E) is beneficial, replacing with the acidicresidue aspartate (i.e. K139D) is likely also to be beneficial.Similarly, since replacing the hydrophobic residue valine at position166 with the aromatic residue phenylalanine is beneficial, replacingwith the aromatic residues tyrosine or tryptophan (i.e. V166Y or V166W)are likely also to be beneficial. An advantageous hyperactive Amyeloistransposase comprises an amino acid substitution at one or morepositions selected from amino acid 79, 95, 115, 116, 121, 139, 166, 179,187, 198, 203, 211, 238, 273, 273, 304, 304, 329, 345, 362, 366, 408,416, 435, 458, 475, 483, 491, 529, 540, 560 and 563 for example one ormore substitutions selected from D79N, R95S, L115D, E116P, H121Q, K139E,V166F, G179N, W187F, P198R, L203R, N211R, E238D, L273M, L273I, D304R,D304K, Q329G, T345L, K362R, T366R, L408M, S416E, S435G, L458M, V475I,N483K, I491M, A529P, K540R, S560K and S563K, or analogous changes at thesame positions.

Of the 59 amino acid substitutions selected, only one (G174A) was foundexclusively in essentially inactive transposases. This indicates thatamino acid changes that make the Amyelois transposase sequence closer toa consensus sequence for active piggyBac-like transposases aresubstitutions that can be incorporated the Amyelois transposase tocreate variants that retain transposition and excision activity. Theseare the changes listed in Table 1 column D. The positive results fromthe initial set of substitutions provide evidence that other changes incolumn D of Table 1 can also be incorporated into an Amyeloistransposase to improve its activity.

6.1.6.2 Second Set of Amyelois Transposase Variants

As described in Liao et al (2007, BMC Biotechnology 2007, 7:16doi:10.1186/1472-6750-7-16 “Engineering proteinase K using machinelearning and synthetic genes”), and U.S. Pat. No. 8,635,029, Sections5.4.2 and 5.4.3, substitutions that have been tested several times inthe contexts of different combinations of other substitutions and thathave “a positive regression coefficient, weight or other valuedescribing its relative or absolute contribution to one or moreactivity” of a protein are usefully incorporated into a protein toobtain a protein that is “improved for one or more property, activity orfunction of interest”. Based on the substitution weights shown in Table8, we designed a set of open reading frames encoding 57 new variants(with sequences given by SEQ ID NOs: 114-170) combining some of the mostpositive substitutions (R95S, K139E, V166F, G179N, W187F, P198R, L203R,N211R, D304K, D304R, T345L, T366R, K540R). Each substitution wasrepresented at least 10 times within the set of 57 variants, and thenumber of different pairwise combinations of substitutions was maximizedso that each substitution was tested in as many different sequencecontexts as possible. Each variant open reading frame was cloned into avector comprising a leucine selectable marker; each open reading frameencoding a transposase variant was operably linked to the Saccharomycescerevisiae Gal-1 promoter. Each of these variants was then individuallytransformed into a Saccharomyces cerevisiae strain comprising achromosomally integrated copy of SEQ ID NO: 192, as described in Section6.1.6.1. After 48 hours cells were scraped from the plate into minimalmedia lacking leucine and with galactose as the carbon source. The A600for each culture was adjusted to 2. Cultures were grown for 4 hours ingalactose to induce expression of the transposases, then a5,000×-diluted aliquot was plated on media lacking leucine, uracil andtryptophan (to count transposition) and a 25,000×-diluted aliquot wasplated on media lacking leucine (to count total live cells). Two dayslater, colonies were counted to determine transposition (=number ofcells on -leu-ura-trp media divided by (5×number of cells on -leumedia)) frequencies. The results are shown in Table 9.

In addition to the activities of the 57 new Amyelois transposasevariants, Table 9 also shows the activities of 12 variants from thefirst set that were among the most active variants in that set. Morethan half of the new set of variants had greater transposition activitythan the best variants from the first set, and all of the new variantshad substantially (more than 50-fold) more transposition activity thanthe naturally occurring Amyelois transposase. A preferred hyperactiveAmyelois transposase comprises an amino acid substitution selected fromR95S, K139E, V166F, G179N, W187F, P198R, L203R, N211R, D304K, D304R,T345L, T366R, K540R, or analogous changes at the same positions.

Brief Description of Tables

Table 1. Amino Acid Changes Likely to Result in Enhanced TransposaseActivity.

Amino acid substitutions with the potential to improve transposaseactivity were identified as described in Section 5.2.6. Column A showsthe position in an Amyelois transposase (relative to SEQ ID NO: 18),column B shows the amino acid in the native protein, column C shows theamino acids found in known active piggyBac-like transposases at theequivalent position in an alignment, column D shows amino acid changesfound in known active piggyBac-like transposases other than the Amyeloistransposase at positions where there is good conservation within therest of the transposase set, but the amino acid in the Amyeloistransposase sequence is an outlier. Mutation to these amino acids areparticularly likely to result in enhanced transposase activity. Morethan one amino acid letter in column means that each of those individualamino acid substitutions are acceptable or beneficial, it is notintended to represent a peptide. For example, at position 2, amino acidsT, A, R, D or N are all acceptable, so column C contains “TARDN” toindicate this.

Table 2. Excision and Transposition of Transposons in Yeast.

Transposon and transposase sources are listed in column A. The leftsequence with SEQ ID NO shown in column B and the right sequence withSEQ ID NO shown in column C were used to construct reporter plasmids asdescribed in Section 6.1.2. The reporter plasmids have insert sequencegiven by the SEQ ID NO listed in column D. These reporter plasmids wereintegrated into the Ura3 gene of a Trp-strain of Saccharomycescerevisiae. The amino acid sequence given by the SEQ ID NO shown incolumn E was backtranslated, synthesized and cloned into a plasmidcomprising a Leu2 gene expressible in Saccharomyces cerevisiae and 2micron origin of replication. The transposase gene was operably linkedto a Gall promoter. The plasmid comprising the transposase wastransformed into the reporter strain, expression was induced, and cellswere plated as described in Section 6.1.1. Induced culture was diluted25,000-fold prior to plating 100 μl on leu dropout plates, and 100-foldprior to plating 100 μl on leu ura or leu ura trp dropout plates. ColumnF shows the number of colonies on the leu dropout plates; column G showsthe number of colonies on the leu ura dropout plates (indicatingexcision of the transposon from the middle of the ura gene in thereporter); column H shows the number of colonies on the leu ura trpdropout plates (indicating excision of the transposon from the middle ofthe ura gene in the reporter and transposition to another site in thegenome).

Table 3. Transposition of Transposons into the Genome of CHO TargetCells.

Cells were transfected with transposons and transposases as described inSection 6.1.3. The transposon SEQ ID NO is shown in row 1, theco-transfected transposase SEQ ID NO is shown in row 2. For eachtransfection, viability (the percentage of cells that are viable) andthe total viable cell density (in millions of cells per ml) are shown inadjacent columns, as indicated in row 3. Rows 4-18 show thesemeasurements at various times post-transfection, the days elapsed areshown in column A.

Table 4. Antibody Production from Transposons Integrated into the Genomeof CHO Target Cells.

Cells were transfected with a transposon (with SEQ ID NO: 256) and DNAencoding a transposase with polypeptide sequence given by SEQ ID NO: 18as described in Section 6.1.3. Recovery is shown in Table 3. During a 14day fed batch antibody production run, the culture supernatant containedthe concentration of antibody shown: column A shows the titer on Day 7;column B shows the titer on Day 10; column C shows the titer on Day 12;column D shows the titer on Day 14.

Table 5. Transposition of Transposons into the Genome of CHO TargetCells by mRNA-Encoded Transposase.

Cells were transfected with a transposon with SEQ ID NO: 256 andmRNA-encoding transposase with SEQ ID NO: 18 as described in Section6.1.4. The viability (the percentage of cells that are viable) and thetotal viable cell density (in millions of cells per ml) are shown incolumns B and C respectively, as indicated in row 1. Rows 2-12 showthese measurements at various times post-transfection, the days elapsedsince transfection are shown in column A.

Table 6. Transposition of Transposons with Truncated End Sequences intothe Genome of CHO Target Cells.

Cells were transfected with a transposon and mRNA-encoded transposase asdescribed in Section 6.1.5. The transposon comprised a left transposonend comprising a 5′-TTAA-3′ integration target sequence immediatelyadjacent to a transposon ITR sequence with SEQ ID NO: 11 which wasimmediately adjacent to a left end sequence with SEQ ID NO shown in row2. The transposon further comprised a right transposon end comprising aright end sequence with SEQ ID NO shown in row 3 immediately adjacent toa transposon ITR sequence with SEQ ID NO: 12 which was immediatelyadjacent to a 5′-TTAA-3′ integration target sequence. The two transposonends were placed on either side of a glutamine synthetase reporter withSEQ ID NO: 370. Row 1 shows the SEQ ID NO of the transposon includingthe target sequences, the ITRs, the additional transposon end sequencesand the selectable marker. Row 4 shows the SEQ ID NO of the transposaseencoded by the transfected mRNA. The viability (the percentage of cellsthat are viable) is indicated in columns labelled “V” in row 5 and thetotal viable cell density (in millions of cells per ml) is indicated incolumns labelled “VCD” in row 5. Rows 6-15 show these measurements atvarious times post-transfection, the days elapsed since transfection areshown in column M.

Table 7. Transposition and Excision Activities of Amyelois TransposaseVariants (First Set).

Genes encoding Amyelois transposase variants were designed, synthesizedand cloned as described in Section 6.1.6.1. SEQ ID NOs of each variantare given in column A. Genes were transformed into a Saccharomycescerevisiae strain whose genome comprised a single copy of transposasereporter SEQ ID NO: 192 and plated on media lacking leucine. After 48hours cells were scraped from the plate into minimal media lackingleucine and with galactose as the carbon source. The A600 for eachculture was adjusted to 2. Cultures were grown for 4 hours in galactoseto induce expression of the transposases. Cultures were diluted 100-foldinto minimal media lacking leucine; one 100 μl aliquot was plated ontominimal media agar plates lacking leucine and uracil (to measuretransposon excision) another 100 μl aliquot was plated onto minimalmedia agar plates lacking leucine, tryptophan and uracil (to measuretransposon transposition). Each culture was also diluted 25,000-fold anda 100 μl aliquot was plated onto minimal media agar plates lackingleucine (to measure live cells). After 48 hours colonies on each platewere counted, the number of colonies on plates lacking leucine are shownin column B, the number of colonies on plates lacking leucine, uraciland tryptophan are shown in column C, the number of colonies on plateslacking leucine and uracil are shown in column D. Column E shows thetransposition frequency (calculated as the number in column C, dividedby the number in column B, and further divided by 250) Column F showsthe excision frequency (calculated as the number in column D, divided bythe number in column B, and further divided by 250). Asterisks in columnG indicate variants where the colonies on plates lacking leucine anduracil or plates lacking leucine, tryptophan and uracil were too denseto be accurately counted. Re-measurements of these variants are shown inTable 9.

Table 8. Model Weights for Amino Acid Substitutions in AmyeloisTransposase Variants.

The effects of sequence changes on Amyelois transposase excision andtransposition activities were modelled as described in Liao et al (2007,BMC Biotechnology 2007, 7:16 doi:10.1186/1472-6750-7-16 “Engineeringproteinase K using machine learning and synthetic genes”) and U.S. Pat.No. 8,635,029. The mean values and standard deviations for theregression weights were calculated for each substitution. The position(relative to SEQ ID NO: 18) is shown in column A, the amino acid foundat this position in SEQ ID NO: 18 is shown in column B. The tested aminoacid substitution is shown in column C. The regression weight for thesubstitution on transposition activity using a log data transformationis shown in column D, the standard deviation for this regression weightis shown in column E. The regression weight for the substitution ontransposition activity using a log data transformation is shown incolumn F, the standard deviation for this regression weight is shown incolumn G.

Table 9. Transposition Activities of Amyelois Transposase Variants(Second Set).

Genes encoding Amyelois transposase variants were designed, synthesizedand cloned as described in Section 6.1.6.2. SEQ ID NOs of each variantare given in column A. Genes were transformed into a Saccharomycescerevisiae strain whose genome comprised a single copy of transposasereporter SEQ ID NO: 192 and plated on media lacking leucine. After 48hours cells were scraped from the plate into minimal media lackingleucine and with galactose as the carbon source. The A600 for eachculture was adjusted to 2. Cultures were grown for 4 hours in galactoseto induce expression of the transposases. Cultures were diluted5,000-fold into minimal media lacking leucine and one 100 μl aliquot wasplated onto minimal media agar plates lacking leucine, tryptophan anduracil (to measure transposon transposition). Each culture was alsodiluted 25,000-fold and a 100 μl aliquot was plated onto minimal mediaagar plates lacking leucine (to measure live cells). After 48 hourscolonies on each plate were counted, the number of colonies on plateslacking leucine are shown in column B, the number of colonies on plateslacking leucine, uracil and tryptophan are shown in column C. Column Dshows the transposition frequency (calculated as the number in column C,divided by the number in column B, and further divided by 5).

TABLE 1 TABLES A B C D amyelois_position amyelois Acceptable Beneficial1 M 2 A TARDN 3 R SRQFI 4 G SGFRLYE 5 L GLTDSRA 6 T RTANDQ 7 D KDEQH 8 LRLEDHN 9 E SEAIR 10 I ILA 11 N GNLASR 12 Q NQLTAH 13 I VIFLCM 14 L HLFM15 E NEDQA 16 L QLSNE 17 E REDSV 18 D ADSLE 19 V AVEDST 20 E KEYLD 21 NNESVFY 22 D RDIPGS 23 V RVSLEGDY 24 I AIEDS 25 F VFISD 26 D VDES 27 E ED28 S SLY 29 G PGSVE 30 D GDEP 31 E TEAKVP 32 S RSEAT 33 D DS 34 H FHNRCE35 V GVDSC 36 S TSIVD 37 I TIDES TDES 38 R LRDSH LDSH 39 V SVDE 40 EWEFQN 41 S LSQY 42 D DFW 43 T NTSC 44 E EDS 45 E DESQ 46 V SVEA SEA 47 ESEMIFTRA 48 I GIDV 49 P SPD SD 50 T ETYFASP 51 L VLD 52 E EHD 53 PDPSEVL 54 Q DQVNE 55 Q TQVNIME 56 G IGSLET 57 S SPADVY 58 S QSED 59 DSDQRE 60 S ESNDP 61 E SENVD 62 N ENLAPID 63 D EDGN 64 Q QMLV 65 P VPE VE66 L ALQVGD 67 S DSTQ 68 N HNSELA 69 L VLSA 70 A TAGR 71 R ERSDQ ESDQ 72R ERGHN 73 S HSTRAM 74 F SFTY 75 Y FYI 76 K YKCRTS 77 G GS 78 K K 79 DNDG NG 80 N RNKEG 81 T YTIHP 82 I KIVCA 83 W W 84 N ANSGY 85 R CRTPK 86A QASTPN 87 P PKC 88 P LPHGSNQ 89 N SNQRTF 90 P RPST 91 R ARTN 92 V VGSI93 R R 94 T VTL 95 R PRS PS 96 S QSAE 97 E HESIL 98 N NP 99 I IP 100 VIVF IF 101 T QTKR 102 G RGMTSE 103 T TNQVR 104 P NPRA 105 G VGQL 106 VSVP SP 107 K NKT 108 R LRVNT 109 Q TQFMDIG 110 A EACT 111 K DKVRS 112 NDNT 113 A PAVI 114 L KLDVYFS 115 L DLET DET 116 E PEI PI 117 L FLQIYS118 D SDLNEK 119 C ICAF 120 F WF 121 H NHQK NQK 122 L KLI 123 F LF 124 VMVIF 125 N DNST DST 126 E DEQS 127 S ESPAD 128 I IM 129 L LEI 130 SQSRHD 131 V EVID ED 132 I TIM 133 L LV 134 E KEDLT 135 H WHMY 136 T T137 N N 138 H EHLVAS 139 K KEYS EYS 140 I IGMA 141 R IRSE 142 S QSRVLH143 E YEVKRS 144 R RFLQ 145 Q SQRTV 146 G KGNQ 147 K FKLENTS 148 NSNIKPA 149 T DTMLA 150 S KSITPE 151 N DNSR 152 E EAS 153 Y PYEAHFV 154 AEAKSTYH 155 Y LYWFMK 156 S RSKHQ 157 E NESDP 158 T LTI 159 T DTN DN 160L MLQTEI 161 T VTMDACS 162 E ED 163 L LMI 164 R HRNWYK 165 A AR 166 VFVYL FYL 167 I IVF 168 G GA 169 L LI 170 L LT 171 Y LYITV 172 L FLMAI173 A TAM 174 G AG A 175 L VL 176 F FYMRIT 177 K KR 178 S SDA 179 G NGKNK 180 R HRG 181 Q EQLMS 182 N NASL 183 L VLTE 184 Q NQSDK 185 D YDSE186 L LW 187 W FWD FD 188 A ANDTR 189 S TSAR 190 D DETS 191 G GEFLV 192T TNSL 193 G GS 194 I RIV 195 E EPTMD 196 I IRV 197 F FY 198 P RPVS RVS199 M CMAST 200 T VT 201 M M 202 S S 203 L KLR KR 204 R NREDQ 205 R RT206 F FY 207 A LAHEDQY 208 F VFML 209 I IL L 210 V LVSIQ 211 N HNR HR212 C CVFNS 213 L LIM 214 R RH 215 F FM 216 D DN 217 D ND 218 S PSRKT219 D DTSA 220 T DTLIV 221 R RP 222 E EVPD 223 E EGTD 224 R RLQ 225 ARAPK RPK 226 A EASGQK 227 I SIDNHT 228 D D 229 R KRAVN 230 L ILFM 231 AAILTH 232 P APK AK 233 I IVLF 234 R SR 235 Q YQDKPS 236 I IVLM 237 YFYWI FWI 238 E TED TD 239 E KEILSQ 240 F FWL 241 V VIS 242 K GKENHQ 243N NIQRC 244 C CLF 245 K QKPRIA 246 D KDLQAN 247 V IVLNA 248 Y YH 249 TNTVS 250 P VP 251 Y CYGS CGS 252 E EPGSAQ 253 N YNHF 254 L ALVI 255 T TC256 I VI 257 D D 258 E E 259 E MERQS 260 L L 261 V VL L 262 A PAGLS 263F F 264 R RK 265 G G 266 R R 267 C TCL 268 K HKPQL 269 F LF 270 R MR 271Q IQMV 272 Y Y 273 L MLI MI 274 P P 275 N MNS 276 K K 277 P PR 278 A ADS279 K KR 280 Y Y 281 G G 282 I LI 283 K KR 284 I LIF 285 I MIWPLYF 286 ACAMK 287 L LAM 288 V CV 289 D DAE 290 A AS 291 Y NYKAGS 292 T NTS 293 YGYSKF 294 Y Y 295 S FSAMTV 296 L YLWISV 297 N NKDY 298 M CMAGFL 299 EYEQIML 300 I IVP 301 Y Y 302 A TALE 303 G G 304 D RDK RK 305 Q GQSD 306P SPT 307 D DGKQSL 308 G GTL 309 P APND 310 Y GYP GP 311 K LKEVPA 312 VTVKPG 313 S ESNC 314 N ENQP 315 K VKLRT 316 P PGSA 317 H THMEGF 318 DQDRFYKE 319 V SVYI 320 V V 321 D IDLKWE 322 R HRED 323 I LIM LM 324 VAVSTI 325 Q KQES 326 P PGT 327 I LIV LV 328 S FSQHLA 329 Q GQR GR 330 TSTQ 331 G NGCH 332 R RHF 333 N NH 334 V IVL 335 T TY 336 M CMVF CVF 337D D 338 N N 339 W WF 340 F FY 341 T TS 342 S SG 343 Y IY I 344 P EPRT345 T LT L 346 Y IYGAFM 347 A EAKTL 348 H YHENA 349 L LM 350 L KLQY 351K KQCN 352 N KNRAEL 353 H HY 354 K GKNDR 355 L LT 356 T TP 357 A CAMIS358 V VLCT 359 G G 360 T T 361 M MVI 362 K KRN RN 363 S KSR 364 N N 365K KR 366 T RTPK RPK 367 C ECGQ EGQ 368 I ILM 369 P P 370 P KPSERD 371 KEKVAS EVAS 372 F FIL 373 R LRKIT 374 E PEKNDR 375 R SRIKT 376 R RN 377 EDEPRQ 378 I VIMGP 379 N GNHEA 380 T ST S 381 S SY 382 L LIMAV 383 F YFL384 G GACR 385 F YFK 386 Q AQTDN 387 D GDEK GEK 388 D QDKPL 389 F NFALINALI 390 T TA 391 I IVL 392 V LVK 393 S SF 394 Y HYF 395 I VICKDA 396 PP 397 K K 398 R KRP 399 N NSAK 400 K KR 401 N ANMV 402 V V 403 F IFLYV404 M LMVA 405 L LM 406 S ST 407 S ST 408 L MLCI MCI 409 H HD 410 H HTED411 D ADNE 412 S ESAN 413 E AESV ASV 414 I VIL 415 D DSNR 416 S ESTQ ETQ417 E TERSQ 418 T TDNR 419 G GDV 420 E EG 421 Q QK 422 Q QMN 423 K K 424P P 425 S ESQDL 426 I IMC MC 427 I IVS 428 T GTLMK 429 F FDYE 430 Y Y431 N NS 432 K KSQ SQ 433 T TY 434 K KM 435 S GSA GA 436 G G 437 V V 438D D 439 N ENSTRV 440 V IVLFT 441 D D 442 K KQE QE 443 L KLVM 444 I CITQS445 R ARKSH 446 T ITSVYN 447 Y YM 448 D TDSN TSN 449 V SVCA 450 S SQNT451 R R 452 N RNK RK 453 S TS 454 R RANK 455 R RA 456 W W 457 P PY 458 LMLK M 459 T VTAK 460 I VIL 461 F FLG 462 F YFI 463 W RWNGY 464 I MILV465 L LVI 466 N DNQ DQ 467 T ITVM IVM 468 A SA 469 G TGAFCLS 470 I VIYR471 N N 472 A SA S 473 K HKYFC 474 I LIV 475 V IVL IL 476 Q YQW YW 477 MDMSCKRQ 478 L ILEHAT 479 N HNIA 480 S HSNKV 481 S SQIPN 482 D DENSG 483N KNVA KVA 484 T TLPV 485 P TPYIQSV 486 T ETRNSYK 487 R RY 488 R GRKT489 A MALEKY 490 F FQ 491 I LIM ML 492 K KERQ 493 K QKENIS 494 L L 495 GAGSYP 496 M RMKTIAL 497 S TSADQL 498 L LM 499 I VITF 500 A LATSGY 501 PPSGE 502 H QHKWFV 503 Q MQIEL 504 A KAQREH 505 E REKQS 506 R RT 507 KAKLVN 508 T LTRKQEP 509 N NPAEK 510 S ESAPMK 511 K RKTPN 512 I LISP 513P PKS 514 V RVADTF 515 S ESATYNH 516 L LAVI 517 R RA 518 K LKVDRQ 519 RSRINL 520 I LIE 521 G AGKTSE 522 S RSINK 523 H VHKIQ 524 L LF 525 GGRKPI 526 E PETNKD 527 S DSVETP 528 S MSNVLT 529 A PAQ P 530 S VSATR 531P PMSH 532 A DAEGV 533 K PKND 534 I QIVNSM 535 P EPDSTR 536 N VNTE 537 VFVPM 538 G KG 539 V TVKPR 540 K RKQY RQY 541 K RKSTV 542 R RYG 543 C C544 Y HYQGTKR 545 I TIVFYDE 546 C C 547 P PSR 548 V LVSYKN 549 K KR 550K LKDI 551 D QDR QR 552 R RS 553 K KMD 554 S STA 555 K TKSNR 556 Y HYTAR557 I TISQY 558 C CF 559 I YIVCKPN 560 S TSKA TKA 561 C C 562 T KTPA KPA563 S KSNR KNR 564 H HFAVNP 565 I VIL 566 C C 567 L LRGFM 568 E QEK 569H CHP 570 A ATNC 571 N KNVIF 572 F QFTDE 573 V VFMIL 574 C CY 575 EAEPQH 576 N DNST 577 C CQ 578 R VRGIFLA

TABLE 2 B C D E A Tposon Tposon Tposon Tpase F G H Source left end rightend SEQ ID NO SEQ ID leu leu ura leu ura trp Amyelois transitella 1 3192 18 454 435 201 Spodoptera litura 216 217 196 172 >250 0 0 Pierisrapae 218 219 197 173 >250 0 0 Myzus persicae 220 221 198 174 >250 0 0Onthophagus taurus 222 223 199 175 >250 0 0 Temnothorax curvispinosus224 225 200 176 >250 0 0 Agrilus planipenn 226 227 201 177 >250 0 0Parasteatoda tepidariorum 228 229 202 178 >250 0 0 Pectinophoragossypiella 230 231 203 179 >250 0 0 Ctenopusia agnata 232 233 204180 >250 0 0 Macrostomum lignano 234 235 205 181 >250 0 0 Orussusabietinus 236 237 206 182 >250 0 0 Eufriesea mexicana 238 239 207 183323 0 0 Spodoptera litura 240 241 208 184 400 0 0 Vanessa tameamea 242243 209 185 389 0 0 Blattella germanica 244 245 210 186 248 0 0Onthophagus taurus 246 247 211 187 >250 0 0 Onthophagus taurus 248 249212 188 >250 0 0 Onthophagus taurus 250 251 213 189 >250 0 0 Megachilerotundata 252 253 214 190 >250 0 0 Xiphophorus maculatus 254 255 215191 >250 0 0

TABLE 3 A B C D E 1 Transposon SEQ ID NO 256 256 256 256 2 TransposaseSEQ ID NO none none 18 18 3 Day viability viable cells viability viablecells 4 1 94.12 1.03 88.76 0.85 5 3 92.15 0.55 93.02 0.21 6 5 80.66 0.2277.66 0.26 7 7 57.58 0.05 50.79 0.06 8 10 27.18 0.03 56.25 0.08 9 1227.05 0.04 75.82 0.25 11 14 31.88 0.04 88.87 1.12 12 17 41.46 0.04 97.563.38 14 19 no live cells no live cells 98.74 >3 15 21 no live cells nolive cells 99.09 >3 16 24 no live cells no live cells >99 >3 17 26 nolive cells no live cells >99 >3 18 27 no live cells no live cells >99 >3

TABLE 4 A B C D Day 7 Day 10 Day 12 Day 14 1,130 1,823 1,932 1,974

TABLE 5 A B C 1 Days post viability viable cells 2 1 94.81 0.78 3 292.41 0.20 4 5 80.89 0.23 5 7 44.26 0.06 6 9 26.54 0.05 7 14 40.70 0.078 16 39.77 0.11 9 19 57.91 0.26 10  21 68.62 0.43 11  23 74.54 0.66 12 26 92.75 1.23

TABLE 6 A B C D E F G H I J K L M 1 Transposon SEQ ID NO 193 193 193 193194 194 194 194 195 195 195 195 2 Left end SEQ ID NO 7 7 7 7 13 13 13 137 7 7 7 3 Right end SEQ ID NO 8 8 8 8 8 8 8 8 16 16 16 16 4 TransposaseSEQ ID NO none none 18 18 none none 18 18 none none 18 18 5 V VCD V VCDV VCD V VCD V VCD V VCD Day 6 98.3 3.807 99.0 3.299 98.3 3.481 99.03.550 98.6 3.781 98.8 2.961 2 7 97.1 0.210 95.0 0.255 91.8 0.151 90.70.114 97.8 0.138 95.5 0.371 4 8 57.8 0.067 64.0 0.101 66.4 0.066 59.10.073 69.6 0.078 70.2 0.180 8 9 31.5 0.026 36.2 0.050 31.4 0.029 38.80.028 39.1 0.047 37.9 0.069 11 10 24.5 0.013 34.0 0.032 20.0 0.011 25.20.026 17.9 0.005 23.9 0.041 13 11 16.8 0.017 39.6 0.095 17.5 0.015 32.40.021 8.5 0.009 15.1 0.025 18 12 15.5 0.017 62.0 0.201 15.5 0.010 59.20.093 12.9 0.014 26.7 0.052 20 13 12.0 0.010 76.9 0.432 16.5 0.014 74.80.164 19.8 0.025 34.9 0.082 21 14 20.3 0.013 97.2 4.714 20.5 0.015 98.34.347 17.8 0.018 94.7 0.832 25 15 14.3 0.011 94.5 1.296 10.3 0.008 99.49.460 1.4 0.001 99.4 5.123 29

TABLE 7 A B C D E F SEQ ID NO live trans ex trans freq ex freq G 36 4360 2 0.0000 0.0000 37 662 22 20 0.0001 0.0001 38 379 1 5 0.0000 0.0001 39581 4 24 0.0000 0.0002 40 411 3 21 0.0000 0.0002 41 269 3 3 0.00000.0000 42 338 10 6 0.0001 0.0001 43 305 4 9 0.0001 0.0001 44 439 9 50.0001 0.0000 45 269 3 7 0.0000 0.0001 46 158 2 0 0.0001 0.0000 47 203 713 0.0001 0.0003 48 273 1 1 0.0000 0.0000 49 205 4 6 0.0001 0.0001 50212 4 7 0.0001 0.0001 96 353 1200 1133 0.0136 0.0128 * 97 521 1200 11220.0092 0.0086 * 98 336 499 568 0.0059 0.0068 * 99 513 900 933 0.00700.0073 * 100 367 511 580 0.0056 0.0063 * 101 237 274 427 0.0046 0.0072102 189 336 285 0.0071 0.0060 103 295 239 367 0.0032 0.0050 104 321 561879 0.0070 0.0110 * 105 181 169 193 0.0037 0.0043 106 275 340 241 0.00490.0035 107 221 610 693 0.0110 0.0125 * 108 204 487 583 0.0095 0.0114 *109 156 134 176 0.0034 0.0045 110 138 299 460 0.0087 0.0133 111 240 220281 0.0037 0.0047 112 227 520 554 0.0092 0.0098 * 113 365 1220 9340.0134 0.0102 * 19 464 224 329 0.0019 0.0028 20 332 153 197 0.00180.0024 21 408 180 102 0.0018 0.0010 22 323 157 117 0.0019 0.0014 23 181135 218 0.0030 0.0048 24 470 255 295 0.0022 0.0025 25 355 185 291 0.00210.0033 26 381 209 207 0.0022 0.0022 27 281 205 183 0.0029 0.0026 28 401274 333 0.0027 0.0033 29 332 201 277 0.0024 0.0033 30 284 137 137 0.00190.0019 31 222 101 110 0.0018 0.0020 32 326 153 158 0.0019 0.0019 33 298189 272 0.0025 0.0037 34 219 111 164 0.0020 0.0030 35 323 248 285 0.00310.0035 51 387 33 39 0.0003 0.0004 52 381 63 83 0.0007 0.0009 53 371 1632 0.0002 0.0003 54 309 38 18 0.0005 0.0002 55 364 142 162 0.0016 0.001856 349 101 87 0.0012 0.0010 57 433 28 16 0.0003 0.0001 58 427 112 1290.0010 0.0012 59 419 19 16 0.0002 0.0002 60 420 92 91 0.0009 0.0009 61629 193 181 0.0012 0.0012 62 308 26 42 0.0003 0.0005 63 225 22 28 0.00040.0005 64 226 63 46 0.0011 0.0008 65 159 28 45 0.0007 0.0011 66 520 121162 0.0009 0.0012 67 351 69 131 0.0008 0.0015 68 173 26 29 0.0006 0.000769 484 149 223 0.0012 0.0018 70 686 28 24 0.0002 0.0001 71 219 35 430.0006 0.0008 72 198 43 64 0.0009 0.0013 73 358 43 33 0.0005 0.0004 74470 17 29 0.0001 0.0002 75 306 41 82 0.0005 0.0011 76 380 127 138 0.00130.0015 77 289 37 33 0.0005 0.0005 78 271 93 145 0.0014 0.0021 79 445 187202 0.0017 0.0018 80 393 21 32 0.0002 0.0003 81 179 17 15 0.0004 0.000382 259 71 91 0.0011 0.0014 83 287 68 74 0.0009 0.0010 84 233 49 620.0008 0.0011 85 255 61 83 0.0010 0.0013 86 247 20 13 0.0003 0.0002 87233 25 28 0.0004 0.0005 88 203 76 132 0.0015 0.0026 89 219 61 75 0.00110.0014 90 294 107 140 0.0015 0.0019 91 219 72 99 0.0013 0.0018 92 220 8171 0.0015 0.0013 93 270 10 29 0.0001 0.0004 94 286 22 31 0.0003 0.000495 255 67 134 0.0011 0.0021 18 247 167 228 0.0027 0.0037

TABLE 8 E G A B C D Weight F Weight Posi- Amino Substi- Weight StdWeight Std tion acid tution method 1 method 1 method 2 method 2 79 D N−0.201 0.094 0.016 0.023 95 R S −0.157 0.090 0.054 0.039 115 L D 0.2740.117 −0.063 0.030 116 E P 0.082 0.098 −0.007 0.022 121 H Q 0.046 0.1060.118 0.049 139 K E 0.450 0.125 0.347 0.055 166 V F 0.388 0.152 0.2300.078 179 G N 0.418 0.111 0.349 0.046 187 W F 0.321 0.102 0.225 0.051198 P R −0.081 0.084 0.116 0.038 203 L R 0.414 0.107 0.050 0.026 211 N R0.251 0.081 0.036 0.025 238 E D −0.107 0.110 0.039 0.052 273 L M 0.0470.090 0.022 0.021 273 L I −0.081 0.097 0.017 0.047 304 D R 0.506 0.0840.319 0.047 304 D K 0.451 0.083 0.289 0.046 329 Q G 0.064 0.089 −0.0300.024 345 T L 0.224 0.088 0.092 0.043 362 K R 0.004 0.083 0.008 0.027366 T R 0.200 0.093 −0.026 0.028 408 L M −0.119 0.111 0.094 0.047 416 SE 0.010 0.130 −0.057 0.027 435 S G 0.012 0.095 0.102 0.028 458 L M−0.078 0.177 0.198 0.062 475 V I 0.109 0.123 0.130 0.053 483 N K −0.1130.086 0.075 0.029 491 I M −0.121 0.112 0.010 0.036 529 A P −0.015 0.1010.007 0.033 540 K R 0.229 0.077 0.117 0.034 560 S K −0.142 0.117 0.1100.043 563 S K 0.027 0.098 0.065 0.027 426 I M −0.112 0.086 −0.009 0.021329 Q R −0.036 0.082 −0.033 0.031 100 V I −0.105 0.091 −0.034 0.038 413E S −0.132 0.083 −0.037 0.018 452 N K −0.208 0.106 −0.043 0.035 65 P E−0.064 0.087 −0.046 0.026 562 T K −0.035 0.090 −0.048 0.021 209 I L−0.003 0.097 −0.054 0.026 159 T N −0.245 0.092 −0.063 0.028 551 D R−0.492 0.084 −0.065 0.037 131 V E −0.408 0.177 −0.072 0.064 472 A S−0.117 0.098 −0.074 0.025 380 T S −0.072 0.103 −0.091 0.023 225 A R−0.218 0.096 −0.093 0.034 336 M C −0.443 0.104 −0.096 0.021 323 I L−0.028 0.106 −0.102 0.027 367 C E −0.180 0.079 −0.116 0.021 310 Y G−0.404 0.099 −0.118 0.019 432 K Q −0.098 0.091 −0.129 0.027 261 V L−0.303 0.107 −0.151 0.031 106 V P −0.398 0.069 −0.197 0.025 232 P A−0.400 0.118 −0.215 0.035 343 Y I −0.632 0.074 −0.252 0.029 467 T M−1.152 0.114 −0.293 0.033 442 K Q −0.929 0.103 −0.309 0.036 466 N D−1.027 0.133 −0.319 0.040 174 G A −1.102 0.204 −0.336 0.047

TABLE 9 A B C D SEQ ID NO leu leu ura trp transposition frequency 170123 393 0.639 156 178 449 0.504 159 135 334 0.495 155 216 498 0.461 168118 264 0.447 143 183 408 0.446 160 151 331 0.438 154 458 1003 0.438 127190 383 0.403 167 145 270 0.372 119 173 304 0.351 131 260 450 0.346 140233 391 0.336 158 330 545 0.330 169 251 413 0.329 125 278 434 0.312 164192 294 0.306 165 176 265 0.301 163 231 340 0.294 157 297 436 0.294 162370 532 0.288 135 200 287 0.287 161 240 339 0.283 141 276 382 0.277 148243 322 0.265 133 260 339 0.261 147 215 272 0.253 114 319 398 0.250 137240 294 0.245 153 228 278 0.244 166 300 363 0.242 142 366 441 0.241 97220 262 0.238 124 366 426 0.233 113 312 360 0.231 96 381 439 0.230 152238 271 0.228 134 294 334 0.227 144 226 246 0.218 118 357 387 0.217 132347 364 0.210 146 412 412 0.200 150 321 320 0.199 151 269 263 0.196 136339 320 0.189 115 435 408 0.188 117 324 294 0.181 123 340 307 0.181 116345 302 0.175 145 280 239 0.171 126 483 405 0.168 139 377 314 0.167 120331 272 0.164 138 405 328 0.162 121 436 342 0.157 149 302 230 0.152 104477 338 0.142 129 366 254 0.139 128 321 211 0.131 130 509 282 0.111 99312 169 0.108 122 556 301 0.108 112 179 82 0.092 107 271 107 0.079 108198 59 0.060 98 278 60 0.043 100 318 45 0.028 101 319 34 0.021 102 15616 0.021 28 179 18 0.020 18 223 2 0.002

7. REFERENCES

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes. To the extent the information associated witha citation may change with time, the version in effect at the effectivefiling date of this application is meant, the effective filing datebeing the filing date of the application or priority application inwhich the citation was first mentioned.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled. Unless otherwise apparentfrom the context, any embodiment, aspect, element, feature or step canbe used in combination with any other.

What is claimed is:
 1. A transposon comprising a heterologouspolynucleotide between left and right transposon ends, wherein the leftend comprises SEQ ID NO: 9 and the right end comprises SEQ ID NO: 10,wherein the transposon is transposable by a transposase having thesequence of SEQ ID NO:18.
 2. The transposon of claim 1, furthercomprising a sequence at least 90% identical to SEQ ID NO: 13immediately adjacent to the left transposon end and between the lefttransposon end and the heterologous polynucleotide and a sequence atleast 90% identical to SEQ ID NO: 16 immediately adjacent to the righttransposon end and between the right transposon end and the heterologouspolynucleotide.
 3. The transposon of claim 1, wherein the heterologouspolynucleotide comprises a heterologous promoter that is active ineukaryotic cells.
 4. The transposon of claim 3, wherein the heterologouspolynucleotide comprises an open reading frame operably linked to theheterologous promoter.
 5. The transposon of claim 3, wherein theheterologous promoter comprises a sequence selected from SEQ ID NOS:474-558.
 6. The transposon of claim 3, wherein the heterologouspolynucleotide comprises in operable linkage with the heterologouspromoter, one or more of: i) a nucleic acid encoding a selectablemarker; ii) a nucleic acid encoding a counter-selectable marker; iii) anucleic acid encoding a regulatory protein; and iv) a nucleic acidencoding an inhibitory RNA.
 7. The transposon of claim 1, wherein theheterologous polynucleotide comprises a heterologous enhancer that isactive in eukaryotic cells.
 8. The transposon of claim 7, wherein theheterologous enhancer is selected from SEQ ID NOS: 453-473.
 9. Thetransposon of claim 1, wherein the heterologous polynucleotide comprisesa heterologous intron that is spliceable in eukaryotic cells.
 10. Thetransposon of claim 9, wherein the nucleotide sequence of theheterologous intron is selected from SEQ ID NOS: 561-621.
 11. Thetransposon of claim 1, wherein the heterologous polynucleotide comprisesan insulator sequence.
 12. The transposon of claim 11, wherein thenucleic acid sequence of the insulator is selected from SEQ ID NOS:435-441.
 13. The transposon of claim 1, wherein the heterologouspolynucleotide comprises or encodes a selectable marker.
 14. Thetransposon of claim 13, wherein the selectable marker is selected from aglutamine synthetase enzyme, a dihydrofolate reductase enzyme, apuromycin acetyltransferase enzyme, a blasticidin acetyltransferaseenzyme, a hygromycin B phosphotransferase enzyme, an aminoglycoside3′-phosphotransferase enzyme and a fluorescent protein.
 15. Thetransposon of claim 1, wherein the heterologous polynucleotide comprisestwo open reading frames, each operably liked to a separate promoter. 16.The transposon of claim 15, wherein the heterologous polynucleotidefurther comprises a sequence selected from SEQ ID NOS: 745-928.
 17. Thetransposon of claim 1, wherein the target sequence 5′-TTAA-3′ isimmediately adjacent to each of SEQ ID NOS:9 and 10 distal from theheterologous polynucleotide.
 18. A eukaryotic cell whose genomecomprises a heterologous polynucleotide between left and righttransposon ends, wherein the left transposon end comprises SEQ ID NO: 9and the right transposon end comprises SEQ ID NO: 10, and the transposonis transposable by a transposase having the sequence of SEQ ID NO:18.19. The eukaryotic cell of claim 18, wherein the cell is an animal cell.20. The animal cell of claim 19, wherein the cell is a mammalian cell.21. The mammalian cell of claim 20, wherein the cell is a rodent cell.22. The mammalian cell of claim 20, wherein the cell is a human cell.23. The eukaryotic cell of claim 18, wherein the target sequence5′-TTAA-3′ is immediately adjacent to each of SEQ ID NOS:9 and 10 distalfrom the heterologous polynucleotide.